Method for the preparation of (+)-calanolide A and analogues thereof

ABSTRACT

A method of preparing (+)-calanolide A, 1, a potent HIV reverse transcriptase inhibitor, from chromene 4 is provided. According to the disclosed method, chromene 4 intermediate was subjected to a chlorotitanium-mediated aldol reaction with acetaldehyde to selectively produce (±)-8a. Separation and enzyme-mediated resolution of (±)-8a produced (+)-8a. Cyclization of (+)-8a under neutral Mitsunobu conditions followed by Luche reduction of (+)-7 produced (+)-calanolide A in high yield and enantiomeric purity. The method of the invention has been extended to produce potent antiviral calanolide A analogues.

CROSS-REFERENCE

This is a division of U.S. patent application Ser. No. 08/609,537, filedMar. 1, 1996, which is a continuation-in-part of U.S. patent applicationSer. No. 08/510,213, filed Aug. 2, 1995, which in turn is acontinuation-in-part of U.S. patent application Ser. No. 08/285,655,filed Aug. 3, 1994, now U.S. Pat. No. 5,489,697 issued Feb. 6, 1996.

FIELD OF THE INVENTION

This invention relates to a method for the preparation of (+)-calanolideA, a potent inhibitor of HIV reverse transcriptase, and calanolide Aanalogues. This invention also relates to the use of calanolide Aanalogues for treating or preventing viral infections.

BACKGROUND OF THE INVENTION

Human immunodeficiency virus (HIV), which is also called humanT-lymphotropic virus type III (HTLV-III), lymphadenopathy-associatedvirus (LAV) or AIDS-associated retrovirus (ARV), was first isolated in1982 and has been identified as the etiologic agent of the acquiredimmunodeficiency syndrome (AIDS) and related diseases. Since then,chemotherapy of AIDS has been one of the most challenging scientificendeavors. So far, AZT, ddC, ddI, 3TC and D4T have been approved by FDAand are being clinically used as drugs for the treatment of AIDS andAIDS-related complex. Although these FDA-approved drugs can extend thelife of AIDS patients and improve their quality of life, none of thesedrugs are capable of curing the disease. Bone-marrow toxicity and otherside effects as well as the emergence of drug-resistant viral strainslimit the long-term use of these agents.¹ On the other hand, the numberof AIDS patients worldwide has increased dramatically within the pastdecade and estimates of the reported cases in the very near future alsocontinue to rise dramatically. It is therefore apparent that there is agreat need for other promising drugs having improved selectivity andactivity to combat AIDS.¹ Several approaches including chemicalsynthesis, natural products screening, and biotechnology have beenutilized to identify compounds targeting different stages of HIVreplication for therapeutic intervention.²

Very recently, the screening program at the National Cancer Institutehas discovered a class of remarkably effective anti-HIV naturalproducts, named calanolides, from the rain forest tree Calophyllumlanigerum, with calanolide A, 1, being the most potent compound in thereported series.³ For example, calanolide A demonstrated 100% protectionagainst the cytopathic effects of HIV-1, one of two distinct types ofHIV, down to a concentration of 0.1 μM. This agent also halted HIV-1replication in human T-lymphoblastic cells (CEM-SS) (EC₅₀ =0.1 μM/IC₅₀=20 μM).³ More interestingly and importantly, calanolide A was found tobe active against both the AZT-resistant G-9106 strain of HIV as well asthe pyridinone-resistant A17 virus.³ Thus, the calanolides, known asHIV-1 specific reverse transcriptase inhibitors, represent novelanti-HIV chemotherapeutic agents for drug development.

A natural source of calanolide A is limited.⁴ Consequently, a practicalsynthesis of the natural product must be developed for further study anddevelopment to be carried out on this active and promising series ofcompounds. Herein, we describe, a method for the synthesis of(±)-calanolide A, (+)-calanolide A and calanolide A analogues. ##STR1##

OBJECTS OF THE INVENTION

Accordingly, one object of the present invention is to provide a simpleand practical method for preparing (+)-calanolide A, 1.

Another object of the invention is to provide calanolide A analogues andderivatives and method of preparation thereof.

A further object of the invention is to provide a method for treating orpreventing viral infections using calanolide A analogues andderivatives.

These and other objects of the invention will become apparent in view ofthe detailed description below.

SUMMARY OF THE INVENTION

The present invention relates to methods for the syntheses of(+)-calanolide A and calanolide A analogues, and method of treating orpreventing viral infections using calanolide A analogues andderivatives.

The method of the present invention for preparing (+)-calanolide A, 1,employs chromene 4 as the key intermediate. Chromene 4 is synthesized bythe sequence depicted in Scheme I. Thus, 5,7-dihydroxy-4-propylcoumarin,2,⁵ was prepared quantitatively from ethyl butyrylacetate andphloroglucinol under Pechmann conditions.⁶ Product yield and purity weredependent on the amount of sulfuric acid used. The 8-position of5,7-dihydroxy-4-propylcoumarin, 2, was then selectively acylated at8-10° C. by propionyl chloride and AlCl₃ in a mixture of carbondisulfide and nitrobenzene to afford5,7-dihydroxy-8-propionyl-4-propylcoumarin, 3.

In an alternative and preferred reaction, coumarin intermediate 3 may beproduced in large scale quantities and with minimal formation ofundesirable 6-position acylated product and 6,8-bis-acylated product byselective acylation of 5,7-dihydroxy-4-propylcoumarin 2 with a mixtureof propionic anhydride and AlCl₃ at about 70-75° C.

The chromene ring was introduced upon treatment of compound 3 with4,4-dimethoxy-2-methylbutan-2-ol, providing 4 in 78% yield (Scheme I).Chlorotitanium-mediated aldol reaction of chromene 4 with acetaldehydeled to formation of (±)-8a and (±)-8b in a ratio of 95:5. The racemicsyn aldol product [(±)-8a] was resolved by enzyme-catalyzed acylation.Thus, in the presence of lipase and vinyl acetate, (-)-8a wasselectively acylated and the desired enantiomer (+)-8a was unreacted.The purified (+)-8a was subjected to a Mitsunobu^(7a-c) reaction,exclusively leading to (+)-trans-chromanone [(+)-7].

Finally, Luche reduction⁸ on (+)-7 led to formation of (+)-calanolide A[(+)-1] which contained 10% of (+)-calanolide B (see Scheme III).(+)-Calanolide A [(+)-1] was further separated from (+)-calanolide B bypreparative normal phase HPLC and was identical with an authenticsample.

If desired, the racemic anti aldol product [(+)-8b] may also be resolvedby enzyme-catalyzed acylation into (+)-8b and the ester 10 from (-)-8b(Scheme IV). Mitsunobu reaction on (+)-8b would lead to formation of thecis-chromanone 7a which could then be reduced to produce calanolide C.

The synthetic sequence for (+)-calanolide A was extended to thesynthesis of calanolide analogues. Thus, Pechmann reaction ofphloroglucinol with various β-ketoesters yields substituted5,7-dihydroxycoumarin 11 (Scheme V). Friedel-Crafts acylation ofsubstituted 5,7-dihydroxycoumarin 11 leads to formation of 8-acylated5,7-dihydroxycoumarin 12. Chromenylation of 12 can be achieved byreacting with substituted β-hydroxyaldehyde dimethylacetal, affordingchromenocoumarin 13. Aldol reaction of chromenocoumarin 13 with carbonylcompounds in the presence of LDA with or without metal complexing agentsforms the racemic aldol product (±)-14. Cyclization of (±)-14 underMitsunobu conditions, by using triphenylphosphine and diethylazodicarboxylate (DEAD), leads to formation of chromanone analogue(±)-15. Reduction of (±)-15 with sodium borohydride with or withoutcerium chloride yields the 12-hydroxy analogue (±)-16 (Scheme V).

Catalytic hydrogenation of both (±)-15 and (±)-16 produces 7,8-dihydroderivatives (±)-17 and (±)-18 (Scheme VI). Treatment of (±)-15 withhydroxylamine or alkoxyamine affords oxime derivatives (±)-19 (SchemeVI). Reduction of (±)-19 under different conditions⁹ should selectivelyyield hydroxylamino or amino compounds (20 and 21).

Optically active forms of 14-21 would be obtained by employing enzymaticacylation, as described in Scheme III for (+)-calanolide A [(+)-1].Thus, enzyme-catalyzed acylation of the racemic aldol product (±)-14would selectively acylate one enantiomer [i.e. (-)-14] and leave theother enantiomer [i.e. (+)-14] unreacted, which would be easilyseparated by conventional methods such as silica gel columnchromatography. The acylated enantiomer [i.e. (-)-14] may be hydrolyzedto form the pure enantiomer [i.e. (-)-14]. The optically pureenantiomers thus obtained [(+)-14 and (-)-14] will be cyclized to (+)-15and (-)-15, respectively, by Mitsunobu reaction. Reduction of (+)-15 and(-)-15 would lead to formation of (+)-16 and (-)-16. Hydrogenation ofoptically active forms of 15 and 16 would provide pure enantiomers of 17and 18 [(+)-and (-)-17; (+)-and (-)-18]. Treatment of pure enantiomersof 15 with hydroxylamine and alkoxylamine affords enantiomerically pureoxime 19 [(+)-and (-)-19]. If desired, (+)-19 and (-)-19 may be reducedto produce enantiomerically pure 20 and 21 [(+)-and (-)-20; (+)-and(-)-21].

The 12-hydroxyl group in compound 1, 16, and 17 as well as theiroptically active forms can be epimerized by a number of methodsincluding acidic conditions, neutral Mitsunobu conditions^(7a-c), orwith DAST.^(7d) An example showing conversion of (-)-calanolide A[(-)-1] into (-)-calanolide B is depicted in Scheme VII.

The process of the present invention may be extended to prepare a widevariety of calanolide analogues such as Formulas I-VI shown in SchemeVIII wherein for Formulas I-V, R₁ is H, halogen, hydroxyl, amino, C₁₋₆alkyl, aryl-C₁₋₆ alkyl, mono- or poly- fluorinated C₁₋₆ alkyl,hydroxy-C₁₋₆ alkyl, C₁₋₆ alkoxy, amino-C₁₋₈ alkyl, C₁₋₆ alkylamino,di(C₁₋₆ alkyl)amino, C₁₋₈ alkylamino-C₁₋₈ alkyl, di(C₁₋₆alkyl)amino-C₁₋₈ alkyl, cyclohexyl, aryl or heterocycle, wherein aryl orheterocycle may each be unsubstituted or substituted with one or more ofthe following: C₁₋₆ alkyl, C₁₋₆ alkoxy, hydroxy-C₁₋₄ alkyl, hydroxyl,amino, C₁₋₆ alkylamino, di(C₁₋₆ alkyl)amino, amino-C₁₋₈ alkyl, C₁₋₈alkylamino-C₁₋₈ alkyl, di(C₁₋₆ alkyl)amino-C₁₋₈ alkyl, nitro, azido orhalogen;

R₂ is H, halogen, hydroxyl, C₁₋₆ alkyl, aryl-C₁₋₆ alkyl, mono- or poly-fluorinated C₁₋₆ alkyl, aryl or heterocycle;

R₃ and R₄ are independently selected from the group consisting of H,halogen, hydroxyl, amino, C₁₋₆ alkyl, aryl-C₁₋₆ alkyl, mono- or poly-fluorinated C₁₋₆ alkyl, hydroxy-C₁₋₆ alkyl, amino-C₁₋₈ alkyl, C₁₋₈alkylamino-C₁₋₈ alkyl, di(C₁₋₆ alkyl)amino-C₁₋₈ alkyl, cyclohexyl, arylor heterocycle; and R₃ and R₄ can be taken together to form a 5-7membered saturated cycle ring or heterocyclic ring;

R₅ and R₆ are independently selected from the group consisting of H,C₁₋₆ alkyl, aryl-C₁₋₆ alkyl, mono- or poly-fluorinated C₁₋₆ alkyl, arylor heterocycle; and R₅ and R₆ can be taken together to form a 5-7membered saturated cycle ring or heterocyclic ring;

R₇ is H, halogen, methyl, or ethyl;

R₈ and R₉ are independently selected from the group consisting of H,halogen, C₁₋₆ alkyl, aryl-C₁₋₆ alkyl, mono- or poly- fluorinated C₁₋₆alkyl, amino-C₁₋₈ alkyl, C₁₋₈ alkylamino-C₁₋₈ alkyl,di (C₁₋₆alkyl)amino-C₁₋₈ alkyl, cyclohexyl, aryl or heterocycle; and R₈ and R₉and be taken together to form a 5-7 membered saturated cycle ring orheterocyclic ring.

For Formula II compounds wherein R₁, R₂, R₃, R₄, R₅, R₆, R₇, R₈, and R₉are the same as defined above, R₁₀ is H, acyl, P(O)(OH)₂, S(O)(OH)₂,CO(C₁₋₁₀ alkyl)CO₂ H, (C₁₋₈ alkyl)CO₂ H, CO (C₁₋₁₀ alkyl)NR₁₁ R₁₂, (C₁₋₈alkyl) NR₁₁ R₁₂ ; wherein R₁₁ and R₁₂ are independently selected fromthe group consisting of H, C₁₋₆ alkyl; and R₁₁ and R₁₂ can be takentogether to form a 5-7 membered saturated heterocyclic ring containingsaid nitrogen.

For Formulae III and IV wherein R₁, R₂, R₃, R₄, R₅, R₆, R₇, R₈, and R₉are the same as defined above, R₁₀ is halogen, OR₁₁, NHOR₁₁, NHOR₁₂,NR₁₁ R₁₂, NR₁₂ R₁₃ ; wherein R₁₁ is H, acyl, P(O) (OH)₂, S(O) (OH)₂,CO(C₁₋₁₀ alkyl)CO₂ H, (C₁₋₈ alkyl)CO₂ H, CO(C₁₋₁₀ alkyl)NR₁₂ R₁₃, (C₁₋₈alkyl) NR₁₂ R₁₃ ; R₁₂ and R₁₃ are independently selected from the groupconsisting of H, C₁₋₆ alkyl; and R₁₂ and R₁₃ can be taken together toform a 5-7 membered saturated heterocyclic ring containing saidnitrogen.

For Formula VI compounds, wherein R₁, R₂, R₃, R₄, R₅, R₆, R₇, R₈, and R₉are the same as defined above, R₁₀ is H, C₁₋₆ alkyl, aryl or aryl C₁₋₆alkyl. ##STR2##

DESCRIPTION OF THE DRAWINGS

FIGS. 1(a) to 1(e) illustrate in vitro MTT assay results, as describedin Example 37, using G910-6 HIV viral strain which is AZT-resistant.

FIGS. 2(a) to 2(e) illustrate in vitro MTT assay results, as describedin Example 37, using H112-2 HIV viral strain which was not pre-treatedwith AZT.

FIGS. 3(a) to 3(e) illustrate in vitro MTT assay results, as describedin Example 37, using A-17 HIV viral strain which is resistant tonon-nucleoside inhibitors such as TIBO but is sensitive to AZT.

FIGS. 4(a) to 4(d) illustrate in vitro MTT assay results, as describedin Example 37, using IIIB cultivated HIV viral strain.

FIGS. 5(a) to 5(d) illustrate in vitro MTT assay results, as describedin Example 37, using RF cultivated HIV viral strain.

FIG. 6 is an HPLC chromatogram of (a) (±)-calanolide A on normal phasecolumn; (b) (±)-calanolide A on a chiral HPLC column; (c) (+)-calanolideA on a chiral HPLC column and (d) (-)-calanolide A on a chiral HPLCcolumn. The HPLC conditions are described in Example 13.

FIG. 7 illustrates representative examples of inventive compounds thatwere evaluated in the in vitro MTT assay of Example 38.

DETAILED DESCRIPTION OF THE INVENTION

All patents, patent applications, and literature references cited hereinare incorporated by reference in their entirety.

The present invention relates to methods for the preparation ofoptically pure (+)-calanolide A and calanolide A analogues andhomologues thereof and compounds produced by the inventive method.

In one embodiment of the invention, a process is provided for preparing(+)-calanolide A from chromene 4, a key intermediate, as shown inschemes I and III. According to the method of the present invention,chromene 4 may be prepared from 5,7-dihydroxy-4-propylcoumarin, 2, isshown in Scheme I. According to this synthetic scheme,5,7-dihydroxy-4-propylcoumarin, 2,⁵ was prepared quantitatively fromethyl butyrylacetate and phloroglucinol under Pechmann conditions.⁶

In conducting this reaction, a volume of a concentrated acid is added ina dropwise manner to a stirring mixture of ethyl butyrylacetate andphloroglucinol with a molar ratio ranging between about 3:1 and about1:3, with a preferable range being about 0.9:1.0. The dropwise additionof an acid was conducted at a rate such that the temperature of thereaction mixture is maintained at a temperature ranging between about 0°C. and about 120° C., preferably about 90° C.

Suitable, but not limiting, examples of concentrated acid includesulfuric acid, trifluoroacetic acid, and methanesulfonic acid. Inpracticing this invention, concentrated sulfuric acid is particularlypreferred. As the product yield and purity appear to be dependent on theamount of concentrated sulfuric acid used, it is preferred that theamount of concentrated sulfuric acid ranges between about 0.5 and 10mole, most preferably ranging between about 2 and about 3.5 mole, permole of ethyl butyrylacetate.

The reaction mixture is then heated to a temperature ranging betweenabout 40° C. and about 150° C., preferably about 90° C., until thereaction reaches completion as determined by TLC analysis. The reactionmixture is then poured onto ice and the precipitated product iscollected by filtration and dissolved in an organic solvent. Suitable,but non-limiting, examples of organic solvents include ethyl acetate,chloroform, and tetrahydrofuran. A preferred solvent is ethyl acetate.The resulting solution is then washed with brine and dried over asuitable drying agent, e.g., sodium sulfate. The yields of this reactionare generally quantitative.

Thereafter, 5,7-dihydroxy-8-propionyl-4-propylcoumarin, 3, was preparedby selectively acylating the 8-position of5,7-dihydroxy-4-propylcoumarin, 2, with propionyl chloride in thepresence of a Lewis acid catalyst (Friedal-Crafts acylation). Inconducting this reaction, a solution of propionyl chloride in a suitablesolvent, e.g., carbon disulfide, was added in a dropwise manner to avigorously stirred solution of 5,7-dihydroxy-4-propylcoumarin, 2, aLewis acid and an organic solvent cooled in an ice bath. Dropwiseaddition of propionyl chloride is conducted such that the temperature ofthe reaction mixture is maintained at a temperature ranging between 0°C. and about 30° C., preferably between about 8° C. and 10° C.

In practicing the invention, the amount of propionyl chloride usedgenerally ranges between about 0.5 and about 6 moles, preferably rangingbetween about 1 and about 2 moles, per mole of5,7-dihydroxy-4-propylcoumarin, 2.

Non-limiting examples of Lewis acid catalysts useful in the acylationreaction include AlCl₃, BF₃, SnCl₄, ZnCl₂, POCl₃ and TiCl₄. A preferredLewis acid catalyst is AlCl₃. The amount of Lewis acid catalyst relativeto 5,7-dihydroxy-4-propylcoumarin, 2, ranges between about 0.5 and about12 moles, preferably ranging between about 2 and about 5 moles, per moleof 5,7-dihydroxy-4-propylcoumarin, 2.

Non-limiting examples of organic solvent for use in preparing the5,7-dihydroxy-4-propylcoumarin, 2, solution include nitrobenzene,nitromethane, chlorobenzene, or toluene and mixtures thereof. Apreferred organic solvent for use in this invention is nitrobenzene.

Upon completion of the addition of propionyl chloride, the vigorouslystirred reaction mixture is maintained at a temperature ranging betweenabout 0° C. and about 120° C., preferably ranging between about 25° C.and 80° C., until the reaction reaches completion as monitored byconventional means such as TLC analysis. The reaction mixture is thenpoured onto ice and extracted several times with a suitable solvent suchas ethyl acetate, chloroform, methylene chloride, tetrahydrofuran, or amixture of chloroform/methanol. A preferred solvent for this extractionis ethyl acetate. The extracts are then dried over a suitable dryingagent, e.g., sodium sulfate, and the product may be purified byconventional means such as silica gel column chromatography.

On small scale (<1 gram), the yield of5,7-dihydroxy-8-propionyl-4-propylcoumarin 3, produced by the abovedescribed reaction is generally quantitative. However, on larger scale(>1 gram), the reaction was very difficult to control and did notexclusively afford the desired product as the desired 8-positionacylated product 3 was accompanied by the formation of undesired6-position acylated product and 6,8-bis-acylated product. Thus, analternative and preferred route for preparing5,7-dihydroxy-8-propionyl-4-propylcoumarin 3 in large scale quantitieswas devised.

Preparation of 8-acylated coumarin 3 on a 5 gram scale as a singleproduct (45% yield) has been achieved by adding a mixture of propionicanhydride, a Lewis acid, e.g., AlCl₃, and suitable solvent, e.g.,1,2-dichloroethane, into a vigorously stirring pre-heated mixture ofcoumarin, a Lewis acid, e.g., AlCl₃, and suitable solvent, e.g.,1,2-dichloroethane, at a temperature ranging between about 40 and about160° C., preferably ranging between about 70 and about 75° C. Dropwiseaddition of the propionic anhydride solution is conducted at a rate suchthat the temperature of the reaction mixture is maintained within thedesired temperature range.

The amount of propionic anhydride used in the reaction generally rangesbetween about 0.5 and about 10 moles, preferably ranging between about 1and about 2 moles, per mole of 5,7-dihydroxy-4-propylcoumarin 2.

Non-limiting examples of Lewis acid catalysts useful in the acylationreaction include AlCl₃, BF₃, POCl₃, SnCl₄, ZnCl₂ and TiCl₄. A preferredLewis acid catalyst is AlCl₃. The amount of Lewis acid catalyst relativeto 5,7-dihydroxy-4-propylcoumarin, 2, ranges between about 0.5 and about12 moles, preferably ranging between about 2 and about 4 moles, per moleof 5,7-dihydroxy-4-propylcoumarin, 2.

Suitable but nonlimiting examples of solvents for use in the inventioninclude diglyme, nitromethane, 1, 1, 2, 2-tetrachloroethane, and1,2-dichloroethane (preferred). Upon completion of the addition ofpropionyl anhydride, the vigorously stirred reaction mixture ismaintained at a temperature ranging between about 40° C. and about 160°C., preferably ranging between about 70° C. and 75° C., until thereaction reaches completion as monitored by conventional means such asTLC analysis. The workup procedure is the same as described above.

The product was purified without the use of column chromatography toafford the desired product 3. This procedure has been scaled-up to 1.7kg of coumarin (for details see experimental section) and the yield for8-acylated coumarin 3 was 29% after recrystallization. The yield for8-acylated coumarin 3 may be further improved by changing thepurification processing. For example, the crude product may berecrystallized from solvent(s) other than dioxane, or a simple washingwith an appropriate solvent may lead to product pure enough for the nextreaction step.

Thereafter, chromene 4 was prepared by introducing the chromene ringinto 5,7-dihydroxy-8-propionyl-4-propylcoumarin, 3, using4,4-dimethoxy-2-methylbutan-2-ol. According to the method of the presentinvention, a solution of 5,7-dihydroxy-8-propionyl-4-propylcoumarin, 3,and 4,4-dimethoxy-2-methylbutan-2-ol in a suitable organic solvent inthe presence of a base was reacted at a temperature ranging betweenabout 40° C. and about 180° C., preferably ranging between about 100° C.and about 120° C., until the reaction reached completion as determinedby conventional means such as TLC analysis. Water and methanol formedduring the reaction were removed azeotropically via a Dean-Stark trap.

In practicing this invention, the amount of4,4-dimethoxy-2-methylbutan-2-ol employed in the reaction generallyranges between about 0.5 and about 8 moles, preferably ranging betweenabout 2 and about 4 moles, per mole of5,7-dihydroxy-8-propionyl-4-propylcoumarin 3.

Suitable, but not limiting examples of organic solvents includepyridine, triethylamine, N,N-dimethylformamide (DMF), toluene,tetrahydrofuran (THF) or 1,2-dichloroethane. Suitable, but non-limitingexamples of the bases include pyridine, 4-dimethylaminopyridine,triethylamine, N,N-diethylaniline, 1,5-diazabicyclo-[4,3,0]-non-5-ene(DBN), 1,8-diazabicyclo-[5,4,0] undec-7-ene (DBU), sodium carbonate andsodium bicarbonate. Pyridine was used as both base and solvent in thisinvention on a small scale; for scale-up, however, pyridine was used asa base and toluene was used as a solvent.

Upon completion of the reaction, the solvent is removed under reducedpressure and the reaction product is dissolved in a suitable solvent,e.g., ethyl acetate. The solution is then washed sequentially with waterand brine and dried over a suitable drying agent, e.g., sodium sulfate.Thereafter, the crude chromene 4 product can be purified by conventionalmeans such as silica gel column chromatography using 25% ethylacetate/hexane as the elution solvent. The yields of chromene 4generally fall with the range of about 60% and about 85%, usuallyresulting in about 78% yield. Chromene 4 was then used to preparechromanone 7.

A number of alternative routes were devised for preparing chromanone 7from chromene 4 in large scale quantities. These routes were describedin U.S. patent application Ser. No. 08/510,213, filed Aug. 2, 1995, thedisclosure which is incorporated herein in its entirety. For instance,U.S. patent application Ser. No. 08/510,213 describes a one-stepreaction process (paraldehyde one-step reaction), shown in Scheme II,and a two-step reaction process (LDA/sulfuric acid process orLDA/Mitsunobu process) for preparing chromanone 7 from chromene 4.Examples of these reactions are provided in the Examples below. In thisinvention, a new route for preparing chromanone 7 from chromene 4 wasdevised, shown in Scheme III, which introduces a chiral resolution stepbetween the two step LDA/Mitsunobu process described in the 08/510,213application and illustrated below. One of the benefits for including theenzyme acylation/resolution step at this stage of the process is that itprovides a more practical and economical means for producing large scaleamounts of chromanone (+)-7, which would lead to formation of(+)-calanolide A after reduction without the subsequent need for chiralHPLC resolution of the racemic calanolide A.

According to Scheme III, (+)-chromanone 7 was prepared by achlorotitanium-mediated aldol condensation reaction of chromene 4 withacetaldehyde which led to formation of aldol products (±)-8a and (±)-8bin a ratio of 95:5, respectively. In conducting the aldol condensationreaction, a solution of LDA was added dropwise to a solution of chromene4 dissolved in a solvent at a temperature ranging between about -78° C.and about 0° C., preferably about -30° C. and about -78° C. Thereafter,a solution of titanium tetrachloride was added dropwise to the stirringreaction mixture. The resulting solution was then warmed to atemperature ranging between about -78° C. and about 40° C., preferablyabout -40° C., and allowed to stir for about 45 minutes to allow fortransmetallation. Thereafter, the solution was recooled to -78° C.

The amount of LDA added per mole of chromene 4 ranged between about 1and about 4 moles, preferably ranging between about 2 and about 3 permole of chromene 4. Dropwise addition LDA is conducted such that thereaction temperature is maintained within the desired range.

The amount of titanium tetrachloride ranges between about 0.5 and about10 moles, preferably ranging between about 2 and about 4 moles per moleof chromene 4.

Suitable, but not limiting examples of solvent include methylenechloride, THF, diethyl ether, dioxane, etc.

Acetaldehyde was then added dropwise to the reaction mixture in amountsranging between about 1 and about 12 moles, preferably ranging betweenabout 4 and about 6 moles per mole of chromene 4. Dropwise addition ofacetaldehyde is conducted such that the reaction temperature ismaintained within the aforementioned range. The reaction was monitoredby conventional means, e.g., TLC analysis, until it reached completion.

The aldol reaction of chromene 4 with acetaldehyde may be carried outunder conditions which employs bases other than LDA. For example, metalhydroxides such as NaOH, KOH and Ca(OH)₂, metal alkoxides such as MeONa,EtONa and t-BuOK, and amines such as pyrrolidine, piperidine,diisopropylethylamine, 1,5-diazabicyclo[4,3,0] non-5-ene (DBN),1,8-diazabicyclo [5,4,0] undec-7-ene (DBU), NaNH₂ and LiHMDS as well ashydrides such as NaH and KH can all be employed for the aldolreactions.¹⁰ Also, aldol reactions can be mediated by metal complexes ofAl, B, Mg, Sn, Zn, Zr and other Ti compounds such as (i-PrO)₃ TiCl,(i-PrO)₄ Ti, PhBCl₂, (n-Bu)₂ BCl, BF₃, (n-Bu)₃ SnCl, SnCl₄, ZnCl₂,MgBr₂, Et₂ AlCl with or without chiral auxiliaries such as1,1'-binaphthol, norephedrinesulfonate, camphanediol, diacetone glucoseand dialkyl tartrate.¹¹⁻¹³

Thereafter, the reaction mixture was quenched at -30° C. to -10° C. withsaturated aqueous ammonium chloride solution and extracted with asuitable solvent, e.g., ethyl acetate. The pooled extracts were washedwith brine and dried over a suitable drying agent, e.g., sodium sulfate.The yields of aldol product generally range between about 40% and about80%, usually about 70%.

It should be noted that the aldol reaction of chromene 4 results in aproduct having two asymmetric centers which in turn would result in adiastereomeric mixture of two sets of enantiomers (four optically activeforms). The mixture may be separated by conventional means to produceracemic syn aldol product (±)-8a and racemic anti aldol product (±)-8bwhich may be resolved into optically active forms. Conventionalresolution methods may be used such as chromatography or fractionalcrystallization of suitable diastereoisomeric derivatives such as saltsor esters with optically active acids (e.g., camphor-10-sulfonic acid,camphoric acid, methoxyacetic acid, or dibenzoyltartaric acid) orenzymatically catalyzed acylation or hydrolysis of the racemic esters.The resultant or synthetic enantiomer may then be transformed toenantioselective synthesis of (+)-calanolide A and its congeners.

In one method, the racemic aldol product may be resolved by highperformance liquid chromatography (HPLC) with organic solvent system asa mobile phase. HPLC is performed on a column packed with chiral packingmaterial. Suitable, but not limiting, examples of chiral packingmaterial include amylose carbamate, D-phenylglycine, L-phenylglycine,D-leucine, L-leucine, D-naphthylalanine, L-naphthylalanine, orL-naphthylleucine. These materials may be bounded, either ionically orcovalently, to silica sphere which particle sizes ranging between about5 μm and about 20 μm. Suitable, but non-limiting, mobile phase includeshexane, heptane, cyclohexane, ethyl acetate, methanol, ethanol, orisopropanol and mixtures thereof. The mobile phase may be employed inisocratic, step gradient or continuous gradient systems at flow ratesgenerally ranging between about 0.5 mL/min. and about 50 mL/min.

In practicing this invention, the racemic product, i.e., syn aldolproduct [(±)-8a], is resolved preferably by enzyme-catalyzed acylation.Enzymatic resolution may employ enzymes such as lipase CC (Candidacylindracea), lipase AK (Candida cylindracea), lipase AY (Candidacylindracea), lipase PS (Pseudomonas Species), lipase AP (Aspergillusniger), lipase N (Rhizopus nieveuis), lipase FAP (Rhizopus nieveus),lipase PP (Porcine Pancrease), pig (porcine) liver esterase (PLE), pigliver acetone powder (PLAP), or subtilisin. Immobilized forms of theenzyme on cellite, molecular sieves, or ion exchange resin are alsocontemplated for use in this method. The amount of enzyme used in thereaction depends on the rate of chemical conversion desired and theactivity of the enzyme. The preferred enzyme for use in theenzyme-catalyzed acylation reaction is lipase.

The enzymatic acylation reaction is carried out in the presence of anacylating agent. Suitable, but not limiting, examples of acylatingagents include vinyl acetate, vinyl propionate, vinyl butyrate, vinylstearate, acetic anhydride, propionic anhydride, phthalic anhydride,acetic acid, propionic acid, hexanoic acid or octanoic acid. Theenzymatic reaction employs at least one mole of acylating agent per moleof aldol product. Acylating agent can be used as a solvent in theacylation reaction or in solution with another solvent such as hexanes,chloroform, benzene, tert-butylmethyl ether, and THF. The preferredsolvent and acylating agent for use in the enzyme-catalyzed acylationare tert-butylmethyl ether and vinyl acetate, respectively.

Suitable, but not limiting examples of solvents for use in the enzymatichydrolysis reaction include water, suitable aqueous buffers such assodium phosphate buffers, or alcohols such as methanol or ethanol.

One skilled in the art will appreciate that racemic esters of aldolproducts can be made by conventional esterification means andselectively hydrolyzed by enzymes so as to produce, in high enantiomericexcess, optically active aldol product, i.e., (+)-8, in free oresterified form.

The purified (+)-8a was subjected to a neutral Mitsunobu reaction,selectively leading to (+)-trans-chromanone[(+)-7]. In performing thisreaction, diethyl azodicarboxylate (DEAD) was added dropwise to asolution containing (+)-8a and triphenylphosphine at a temperatureranging between about -10° C. and about 40° C., preferably about ambienttemperature. The amount of DEAD used in the reaction generally rangesbetween about 1 mole and about 10 moles preferably about 1 mole andabout 4 moles, per mole of aldol (+)-8a. The amount oftriphenylphosphine used in the reaction generally ranged between about 1mole and about 10 moles, preferably ranging between about 1 mole andabout 4 moles, per mole of aldol (+)-8a.

Instead of DEAD, other suitable azo reagents reported in the literaturecan be employed such as diisopropyl azodicarboxylate (DIAD), dibutylazodicarboxylate (DBAD), dipiperidinoazodicarboxamide, bis(N⁴-methylpiperazin-1-yl) azodicarboxamide, dimorpholinoazodicarboxamide,N,N,N',N'-tetramethylazodicarboxamide (TMAD)¹⁴. Also, in addition totriphenylphosphine, other phosphine derivatives such astri-n-butylphosphine,¹⁴ triethylphosphine, trimethylphosphine andtris(dimethylamino)phosphine may be used.

Thereafter, the reaction was quenched with saturated ammonium chlorideupon completion and extracted with a suitable solvent, e.g., ethylacetate. The pooled organic layers were washed with brine, concentratedin vacuo and the crude chromanone (+)-7 was purified by conventionalmeans as discussed above. The yields of chromanone (+)-7 from theMitsunobu reaction generally range between about 60% and about 80%,usually about 70%.

Finally, mild borohydride reduction of chromanone (+)-7 in the presenceof CeCl₃ (H₂ O)₇ (Luche reduction) produced (+)-calanolide A with thedesired stereochemical arrangement. In conducting the reductionreaction, a solution of chromanone (+)-7 was added dropwise into asolution of reducing agent, e.g., sodium borohydride and a metaladditive, e.g., CeCl₃ (H₂ O)₇ in ethanol. The rate of addition is suchthat the reaction mixture temperature is maintained within a range ofbetween about -40° C. and about 60° C., preferably ranging between about-10° C. and about -30° C. Thereafter, the reaction mixture was stirredat a temperature ranging between about -40° C. and about 60° C.

In general, the amount of metal additive, e.g., CeCl₃ (H₂ O)₇ present inthe reaction mixture ranged between about 0.1 and about 2 moles,preferably ranging between bout 0.5 and about 1 mole, per mole of sodiumborohydride. In addition, the amount of reducing agent, e.g., sodiumborohydride employed in the reaction generally ranged between about 0.1and about 12 moles, preferably ranging between about 2 and about 4moles, per mole of chromanone (+)-7. Suitable, but non-limiting,examples of reducing agents include NaBH₄ LiAlH₄,(i-Bu)₂ AlH,(n-Bu)₃SnH,9-BBN, Zn(BH₄)₂, BH₃, DIP-chloride, selectrides and enzymes such asbaker yeast. Suitable, but non-limiting, examples of metal additivesinclude CeCl₃, ZnCl₂, AlCl₃, TiCl₄, SnCl₃, and LnCl₃ and their mixturewith triphenylphosphine oxide. In practicing this invention, sodiumborohydride as reducing agent and CeCl₃ (H₂ O)₇ as metal additive arepreferred.

Thereafter, the reduction mixture was diluted with water and extractedwith a suitable solvent, e.g., ethyl acetate. The extract was dried overa suitable drying agent, e.g., sodium sulfate, and concentrated. Theresulting residue was then purified by conventional means such as silicagel chromatography, using ethyl acetate/hexane solvent mixtures. Luchereduction on (+)-7 led to formation of (+)-calanolide A [(+)-1] whichcontained 10% of (+)-calanolide B. (+)-Calanolide A [(+)-1] was furtherseparated from (+)-calanolide B by preparative normal phase HPLC and wasidentical with an authentic sample.

Thus, (+)-calanolide A, 1, was successfully prepared with the desiredstereochemical arrangement by treatment of the key intermediate chromene4 with chlorotitanium catalyzed aldol reaction to produce (±)-8a, enzymeresolution of the racemate to produce (+)-8a, and neutral Mitsunobureaction of (+)-8a to produce chromanone (+)-7, followed by Luchereduction via chromanone (+)-7 (see Scheme III).

Enzyme resolution of trans-(±)-8b racemate with vinyl acetate and lipaseallowed for the separation of (+)-8b, which, following treatment underneutral Mitsunobu reaction with triphenylphosphine and DEAD andsubsequent Luche reduction, would result in calanolide C (Scheme IV).

In another embodiment of the invention, analogues of calanolide A areprovided by extension of the aforementioned synthetic sequence for(+)-calanolide A. Pechmann reaction of phloroglucinol with substitutedβ-ketoesters yields substituted 5,7-dihydroxycoumarin 11 as shown inScheme V. The conditions and reagents used in the Pechmann reaction aredescribed above.

Suitable, but non-limiting, β-ketoesters include those of formula i:##STR3## wherein R₁ is H, halogen, hydroxyl, amino, C₁₋₆ alkyl,aryl-C₁₋₆ alkyl, mono- or poly- fluorinated C₁₋₆ alkyl, hydroxy-C₁₋₆alkyl, C₁₋₆ alkoxy, amino-C₁₋₈ alkyl, C₁₋₆ alkylamino, di(C₁₋₆alkyl)amino, C₁₋₈ alkylamino-C₁₋₈ alkyl, di(C₁₋₆ alkyl) amino-C₁₋₈alkyl, cyclohexyl, aryl, or heterocycle, wherein aryl or heterocycle mayeach be unsubstituted or substituted with one or more of the following:C₁₋₆ alkyl, C₁₋₆ alkoxy,hydroxy-C₁₋₄ alkyl, hydroxyl, amino, C₁₋₆alkylamino, di(C₁₋₆ alkyl)amino, amino-C₁₋₈ alkyl, C₁₋₈ alkylamino-C₁₋₈alkyl, di(C₁₋₆ alkyl-amino-C₁₋₈ alkyl, nitro, azido or halogene; and R₂is H, halogen, hydroxyl, C₁₋₆ alkyl, aryl-C₁₋₆ alkyl, mono- orpoly-fluorinated C₁₋₆ alkyl, aryl or heterocycle.

Friedel-Crafts acylation of substituted 5,7-dihydroxycoumarin 11 leadsto formation of 8-acylated 5,7-dihydroxycoumarin 12. The conditions andreagents used in the Friedel-Crafts acylation reaction are describedabove.

Non-limiting examples of carboxylic acid anhydrides and halides includeformula ii carboxylic acid anhydrides and halides: ##STR4## wherein X ishalogen (e.g. chloro) or OCOCHR₃ R₄ wherein R₃ and R₄ are independentlyselected from the group consisting of H, halogen, hydroxyl, C₁₋₆ alkyl,aryl-C₁₋₆ alkyl, mono- or poly- fluorinated C₁₋₆ alkyl, hydroxy-C₁₋₆alkyl, amino-C₁₋₈ alkyl, C₁₋₈ alkylamino-C₁₋₈ alkyl, di(C₁₋₆alkyl)amino-C₁₋₈ alkyl, cyclohexyl, aryl or heterocycle; and R₃ and R₄can be taken together to form a 5-7 membered saturated cycle ring orheterocyclic ring.

Chromenylation of 12 can be achieved by reacting with substitutedβ-hydroxyaldehyde dimethylacetal, affording chromenocoumarin 13. Theconditions and amounts of reagents are described above. Representativeexamples of substituted β-hydroxyaldehyde dimethylacetals of formula iiicomprise: ##STR5## wherein R₅ and R₆ are independently selected from thegroup consisting of H, C₁₋₆ alkyl, aryl-C₁₋₆ alkyl, mono- orpoly-fluorinated C₁₋₆ alkyl, aryl or heterocycle; R₅ and R₆ can be takentogether to form a 5-7 membered saturated cycle ring or heterocyclering; and R₇ is H, halogen, methyl, ethyl.

Aldol condensation reaction of chromene 13 with carbonyl compounds inthe presence of LDA forms the racemic aldol product (±)-14. According tothe present invention, a solution of LDA in THF was added dropwise to asolution of chromene 13 in THF at a temperature ranging between about-78° C. and about 0° C., preferably about -30° C. and about -78° C. Theamount of LDA added per mole of chromene 13 ranged between about 1 andabout 4 moles, preferably ranging between about 2 and about 3 moles permole of chromene 13. Dropwise addition of LDA is conducted such that thereaction temperature is maintained within the desired range.

A carbonyl compound of formula iv was then added dropwise to thereaction mixture in amounts ranging between about 1 and about 12 moles,preferably ranging between about 4 and about 6 moles per mole ofchromene 13. Dropwise addition of carbonyl compound is conducted suchthat the reaction temperature is maintained within the aforementionedrange. The reaction was monitored by conventional means, e.g., TLCanalysis, until it reached completion.

Representative examples of formula iv carbonyl compounds comprise:##STR6## wherein R₈ and R₉ are independently selected from the groupconsisting of H, halogen, C₁₋₆ alkyl, aryl-C₁₋₆ alkyl, mono- or poly-fluorinated C₁₋₆ alkyl, hydroxy-C₁₋₆ alkyl, amino-C₁₋₈ alkyl, C₁₋₈alkylamino-C₁₋₈ alkyl, di(C₁₋₆ alkyl)amino-C₁₋₈ alkyl, cyclohexyl, arylor heterocycle; and R₈ and R₉ can be taken together to form a 5-7membered saturated cycle ring or heterocyclic ring.

One skilled in the art will appreciate that the aldol reaction ofchromene 13 with carbonyl compounds of formula iv to form 14 can becarried out under conditions which employs bases other than LDA. Forexample, metal hydroxides such as NaOH, KOH and Ca(OH)₂, metal alkoxidessuch as MeONa, EtONa and t-BuOK, and amines such as pyrrolidine,piperidine, diisopropylethylamine, 1,5-diazabicyclo[4,3,0]non-5-ene(DBN), 1,8-diazabicyclo[5,4,0]undec-7-ene (DBU), NaNH₂ and LiHMDS aswell as hydrides such as NaH and KH can all be employed for the aldolreactions.¹⁰ Also, aldol reactions can be mediated by metal complexes ofAl, B, Mg, Sn, Ti, Zn and Zr compounds such as TiCl₄, (i-PrO)₃ TiCl,(i-PrO)₄ Ti, PhBCl₂, (n-Bu)₂ BCl, BF₃, (n-Bu)₃ SnCl, SnCl₄, ZnCl₂,MgBr₂, Et₂ AlCl with or without chiral auxiliaries such as1,1'-binaphthol, norephedrinesulfonate, camphanediol, diacetone glucoseand dialkyl tartrate.¹¹⁻¹³

Thereafter, the reaction mixture was quenched at -30° C. to -10° C. withsaturated aqueous ammonium chloride solution and extracted with asuitable solvent, e.g., ethyl acetate. The pooled extracts were washedwith brine and dried over a suitable drying agent, e.g., sodium sulfate.The yields of aldol product (±)-14 generally range between about 40% andabout 80%, usually about 70%.

Cyclization of (±)-14 under neutral Mitsunobu conditions, by usingtriphenylphosphine and diethyl azodicarboxylate (DEAD), leads toformation of chromanone analogue (±)-15. Reduction of (±)-15 with sodiumborohydride with or without metal additives such as cerium chlorideyields the 12-hydroxy analogue (±) -16 (Scheme V). The conditions andamounts of reagents used in the Mitsunobu and borohydride reductionreactions are described above.

Catalytic hydrogenation of both (±)-15 and (±)-16 produces 7,8-dihydroderivatives (±)-17 and (±)-18 (Scheme VI). To a solution of (±)-15 or(±)-16 in ethanol or ethanol/methylene chloride mixtures in aconventional Parr apparatus under N₂, hydrogenation catalyst was addedat ambient temperature. The mixture was shaken under hydrogen for a timesufficient to complete the hydrogenation reaction. The solution was thengravity filtered to remove catalyst and solvent was evaporated.

Suitable, but non-limiting, hydrogenation catalysts for use in theinvention include Pd/C, PtO₂ and Rh/C, Raney-Ni. In practicing theinvention, 10% palladium/carbon is preferred. The amount of catalystemployed generally ranges between about 0.01 and about 0.5 mole,preferably ranging between about 0.05 and about 0.1 mole per mole of(±)-15 or (±)-16.

In yet another embodiment of the invention, intermediate chromanones(±)-7, (±)-7, (±)-7a and (±)-15 can be used to prepare oxime,hydroxyamino, alkoxyamino or amino calanolide derivatives. Treatment ofthe said chromanones with hydroxylamine or alkoxyamine affords oximederivatives (±)-19 (Scheme VI).

Representative amines for preparing oxime derivatives comprise NH₂ OR₁₀wherein R₁₀ is H, C₁₋₈ alkyl, phenyl, benzyl, acyl P(O) (OH)₂, S(O)(OH)₂, CO(C₁₋₁₀ alkyl)CO₂ H, (C₁₋₈ alkyl)CO₂ H, CO(C₁₋₁₀ alkyl)NR₁₂ R₁₃,(C₁₋₈ alkyl) NR₁₂ R₁₃ ; wherein R₁₂ and R₁₃ are independently selectedfrom the group consisting of H, C₁₋₆ alkyl; and R₁₂ and R₁₃ can be takentogether to form a 5-7 membered saturated heterocyclic ring containingsaid nitrogen. Examples of useful alkoxyamines include methoxyamine andbenzyloxyamine.

The oxime derivatives may be prepared by refluxing a methanolic solutionof the chromanone with hydroxyl amine or alkoxyamine in the presence ofa metal carbonate such as potassium carbonate or pyridine until thereaction reaches completion. The amount of amine generally rangesbetween about 1 and about 20 moles, preferably between about 3 and about6 moles, per mole of chromanone.

Upon completion of the reaction, filtration of the solution to removesolids and removal of solvent resulted in an oil which was purified viasilica gel chromatography. The yields of oximes generally range betweenabout 30% and about 80%, usually about 50%.

If desired, oxime derivatives (±)-19 may be reduced under differentconditions⁹ to yield hydroxyamino or amino compounds (20 and 21).

Thus, optically active forms of 14-21 (Scheme V and VI) would beobtained by employing enzymatic acylation, as described above, in theprocedure outlined in Scheme III for (±)-calanolide A [(±)-1].Enzyme-catalyzed acylation of the racemic aldol product (±)-14 wouldselectively acylate one enantiomer [i.e. (-)-14] and leave the otherenantiomer [i.e. (+)-14] unreacted, which would be easily separated byconventional methods such as silica gel column chromatography. Theacylated enantiomer [i.e. (-)-14] may be hydrolyzed to form the pureenantiomer [i.e. (-)-14]. The optically pure enantiomers thus obtained[(+)-14 and (-)-14] will be cyclized to (+)-15 and (-)-15, respectively,by the Mitsunobu reaction as described above. Subsequent reduction of(+)-15 and (-)-15 would lead to formation of (+)-16 and (-)-16,respectively. Hydrogenation of optically active forms of 15 and 16 wouldprovide pure enantiomers of 17 and 18 [(+)-and (-)-17; (+)-and (-)-18],respectively. Treatment of pure enantiomers of 15 with hydroxylamine oralkoxyamine, as described above, should afford enantiomerically pureoxime 19 [(+)-and (-)-19]. Reduction of (+)-19 and (-)-19 would lead toformation of enantiomerically pure 20 and 21 [(+)- and (-)-20; (+)-and(-)-21].

The 12-hydroxyl group in compound 1, 16, and 17 as well as theiroptically active forms can be epimerized under a variety of conditionsincluding acidic conditions, neutral Mitsunobu conditions^(7a-c) or withDAST.^(7d) An example showing conversion of (-)-calanolide A [(-)-1]into (-)-calanolide B using DAST^(7d) is depicted in Scheme VII.

Thus, the process of the present invention may be extended to prepare awide variety of calanolide analogues such as Formulas I-VI shown inScheme VIII wherein for Formulas I-V, R₁ is H, halogen, hydroxyl, amino,C₁₋₆ alkyl, aryl-C₁₋₆ alkyl, mono- or poly- fluorinated C₁₋₆ alkyl,hydroxy-C₁₋₆ alkyl, C₁₋₆ alkoxy, amino-C₁₋₈ alkyl, C₁₋₆ alkylamino,di(C₁₋₆ alkyl)amino, C₁₋₈ alkylamino-C₁₋₈ alkyl, di(C₁₋₆alkyl)amino-C₁₋₈ alkyl, cyclohexyl, aryl or heterocycle, wherein aryl orheterocycle may each be unsubstituted or substituted with one or more ofthe following: C₁₋₆ alkyl, C₁₋₆ alkoxy, hydroxy-C₁₋₄ alkyl, hydroxyl,amino, C₁₋₆ alkylamino, di(C₁₋₆ alkyl)amino, amino-C₁₋₈ alkyl, C₁₋₈alkylamino-C₁₋₈ alkyl, di(C₁₋₆ alkyl)amino-C₁₋₈ alkyl, nitro, azido orhalogen;

R₂ is H, halogen, hydroxyl, C₁₋₆ alkyl, aryl-C₁₋₆ alkyl, mono- or poly-fluorinated C₁₋₆ alkyl, aryl or heterocycle;

R₃ and R₄ are independently selected from the group consisting of H,halogen, hydroxyl, amino, C₁₋₆ alkyl, aryl-C₁₋₆ alkyl, mono- or poly-fluorinated C₁₋₆ alkyl, hydroxy-C₁₋₆ alkyl, amino-C₁₋₈ alkyl, C₁₋₈alkylamino-C₁₋₈ alkyl, di(C₁₋₆ alkyl)amino-C₁₋₈ alkyl, cyclohexyl, arylor heterocycle; and R₃ and R₄ can be taken together to form a 5-7membered saturated cycle ring or heterocyclic ring;

R₅ and R₆ are independently selected from the group consisting of H,C₁₋₆ alkyl, aryl-C₁₋₆ alkyl, mono- or poly-fluorinated C₁₋₆ alkyl, arylor heterocycle; and R₅ and R₆ can be taken together to form a 5-7membered saturated cycle ring or heterocyclic ring;

R₇ is H, halogen, methyl, or ethyl;

R₈ and R₉ are independently selected from the group consisting of H,halogen, C₁₋₆ alkyl, aryl-C₁₋₆ alkyl, mono- or poly- fluorinated C₁₋₆alkyl, amino-C₁₋₈ alkyl, C₁₋₈ alkylamino-C₁₋₈ alkyl,di (C₁₋₆ alkyl)amino-C₁₋₈ alkyl, cyclohexyl, aryl or heterocycle; and R₈ and R₉ and betaken together to form a 5-7 membered saturated cycle ring orheterocyclic ring.

For Formula II compounds wherein R₁, R₂, R₃, R₄, R₅, R₆, R₇, R₈, and R₉are the same as defined above, R₁₀ is H, acyl, P(O)(OH)₂, S(O)(OH)₂,CO(C₁₋₁₀ alkyl)CO₂ H, (C₁₋₈ alkyl)CO₂ H, CO(C₁₋₁₀ alkyl)NR₁₁ R₁₂, (C₁₋₈alkyl) NR₁₁ R₁₂ ; wherein R₁₁ and R₁₂ are independently selected fromthe group consisting of H, C₁₋₆ alkyl; and R₁₁ and R₁₂ can be takentogether to form a 5-7 membered saturated heterocyclic ring containingsaid nitrogen.

For Formulae III and IV wherein R₁, R₂, R₃, R₄, R₅, R₆, R₇, R₈, and R₉are the same as defined above, R₁₀ is halogen, OR₁₁, NHOR₁₁, NHOR₁₂,NR₁₁ R₁₂, NR₁₂ R₁₃ ; wherein R₁₁ is H, acyl, P(O)(OH)₂, S(O)(OH)₂,CO(C₁₋₁₀ alkyl)CO₂ H, (C₁₋₈ alkyl)CO₂ H, CO(C₁₋₁₀ alkyl)NR₁₂ R₁₃, (C₁₋₈alkyl) NR₁₂ R₁₃ ; R,₂ and R₁₃ are independently selected from the groupconsisting of H, C₁₋₆ alkyl; and R₁₂ and R₁₃ can be taken together toform a 5-7 membered saturated heterocyclic ring containing saidnitrogen.

For Formula VI compounds, wherein R₁, R₂, R₃, R₄, R₅, R₆, R₇, R₈, and R₉are the same as defined above, R₁₀ is H, C₁₋₆ alkyl, aryl or aryl C₁₋₆alkyl.

In another embodiment of the invention, a method for converting(-)-calanolide A into (-)-calanolide B is provided. It has beendiscovered that (-)-calanolide A may be converted readily to(-)-calanolide B using diethylamidosulfur trifluoride (DAST) or theMitsunobi reaction, e.g., diethyl azodicarboxylate andtriphenylphosphine, under the conditions and ranges described above.

The amount of DAST employed in the inversion reaction generally rangesbetween about 0.5 and about 5.0 moles, preferably ranging between about1 and about 2.0 moles, per mole of (-)-calanolide A. Suitable, butnon-limiting, reaction solvents for use in the invention includemethylene chloride, THF, diethyl ether, or chloroform. In practicing theinvention, the preferred solvent is methylene chloride. The reaction maybe conducted at a temperature ranging between about -78° C. and about50° C., preferably about -78° C., until the reaction is complete asdetermined by usual methods such as thin layer chromatography.

In yet another embodiment of the invention, a method for treating orpreventing viral infections in mammals using calanolide A analogues ispresented. Examples of mammals include humans, primates, bovines,ovines, porcines, felines, canines, etc. Examples of viruses mayinclude, but not be limited to, HIV-1, HIV-2, herpes simplex virus (type1 and 2) (HSV-1 and 2), varicella zoster virus (VZV), cytomegalovirus(CMV), papilloma virus, HTLV-1, HTLV-2, feline leukemia virus (FLV),avian sarcoma viruses such as rous sarcoma virus (RSV), hepatitis typesA-E, equine infections, influenza virus, arboviruses, measles, mumps andrubella viruses. More preferably the compounds of the present inventionwill be used to treat a human infected with a retrovirus. Preferably thecompounds of the present invention will be used to treat a human exposedor infected (i.e., in need of such treatment) with the humanimmunodeficiency virus, either prophylactically or therapeutically. Anadvantage of the compounds of the present invention is that they retainthe ability to inhibit certain HIV RT mutants which are resistant toother non-nucleoside inhibitors such as TIBO and nevirapine or resistantto nucleoside inhibitors. This is advantageous over the current AIDSdrug therapy, where biological resistance often develops to nucleosideanalogs used in the inhibition of RT.

Hence the compounds of the present invention are particularly useful inthe prevention or treatment of infection by the human immunodeficiencyvirus and also in the treatment of consequent pathological conditionsassociated with AIDS. Treating AIDS is defined as including, but notlimited to, treating a wide range of states of HIV infection: AIDS, ARC,both symptomatic and asymptomatic, and actual or potential exposure toHIV. For example, the compounds of this invention are useful in treatinginfection of HIV after suspected exposure to HIV by e.g., bloodtransfusion, exposure to patient blood during surgery or an accidentialneedle stick.

Antiviral calanolide A analogues may be formulated as a solution oflyophilized powders for parenteral administration. Powders may bereconstituted by addition of a suitable diluent or otherpharmaceutically acceptable carrier prior to use. The liquid formulationis generally a buffered, isotonic, aqeuous solution. Examples ofsuitable diluents are normal isotonic saline solution, standard 5%dextrose in water or in buffered sodium or ammonium acetate solution.Such formulation is especially suitable for parenteral administration,but may also be used for oral administration. It may be desirable to addexcipients such as polyvinylpyrrolidone, gelatin, hydroxy cellulose,acacia, polyethylene glycol, mannitol, sodium choride or sodium citrate.

Alternatively, the compounds of the present invention may beencapsulated, tableted or prepared in an emulsion (oil-in-water orwater-in-oil) syrup for oral administration. Pharmaceutically acceptablesolids or liquid carriers, which are generally known in thepharmaceutical formulary arts, may be added to enhance or stabilize thecomposition, or to facilitate preparation of the composition. Solidcarriers include starch (corn or potato), lactose, calcium sulfatedihydrate, terra alba, croscarmellose sodium, magnesium stearate orstearic acid, talc, pectin, acacia, agar, gelatin, maltodextrins andmicrocrystalline cellulose, or collodial silicon dioxide. Liquidcarriers include syrup, peanut oil, olive oil, corn oil, sesame oil,saline and water. The carrier may also include a sustained releasematerial such as glyceryl monostearate or glyceryl distearate, alone orwith a wax. The amount of solid carrier varies but, preferably, will bebetween about 10 mg to about 1 g per dosage unit.

The dosage ranges for administration of antiviral calanolide A analoguesor derivatives are those to produce the desired affect whereby symptomsof infection are ameliorated. For example, as used herein, apharmaceutically effective amount for HIV infection refers to the amountadministered so as to maintain an amount which suppresses or inhibitssecondary infection by syncytia formation or by circulating virusthroughout the period during which HIV infection is evidenced such as bypresence of anti-HIV antibodies, presence of culturable virus andpresence of p24 antigen in patient sera. The presence of anti-HIVantibodies can be determined through use of standard ELISA or Westernblot assays for example, anti-gp120, anti-gp41, anti-tat, anti-p55,anti-p17, antibodies, etc. The dosage will generally vary with age,extent of the infection, the body weight and counterindications, if any,for example, immune tolerance. The dosage will also be determined by theexistence of any adverse side effects that may accompany the compounds.It is always desirable, whenever possible, to keep adverse side effectsto a minimum.

One skilled in the art can easily determine the appropriate dosage,schedule, and method of administration for the exact formulation of thecomposition being used in order to achieve the desired effectiveconcentration in the individual patient. However, the dosage can varyfrom between about 0.001 mg/kg/day to about 50 mg/kg/day, but preferablybetween about 0.01 to about 1.0 mg/kg/day.

The pharmaceutical composition may contain other pharmaceuticals inconjunction with antiviral calanolide A analogues and derivatives totreat (therapeutically or prophylactically) AIDS. For example, otherpharmaceuticals may include, but are not limited to, other antiviralcompounds (e.g., AZT, ddC, ddI, D4T, 3TC, acyclovir, gancyclovir,fluorinated nucleosides and nonnucleoside analog compounds such as TIBOderivatives, nevirapine, saquinavir, α-interfon and recombinant CD4),immunostimulants (e.g., various interleukins and cytokines),immunomodulators and antibiotics (e.g., antibacterial, antifungal,anti-pneumocysitis agents). Administration of the inhibitory compoundswith other anti-retroviral agents that act against other HIV proteinssuch as protease, intergrase and TAT will generally inhibit most or allreplicative stages of the viral life cycle.

In addition, the compounds of the present invention are useful as toolsand/or reagents to study inhibition of retroviral reversetranscriptases. For example, the instant compounds selectively inhibitHIV reverse transcriptase. Hence, the instant compounds are useful as astructure/activity relationship (SAR) tool to study, select and/ordesign other molecules to inhibit HIV.

The following examples are illustrative and do not serve to limit thescope of the invention as claimed.

EXPERIMENTAL

All chemical reagents and solvents referred to herein are readilyavailable from a number of commercial sources including Aldrich ChemicalCo. or Fischer Scientific. NMR spectra were run on a Hitachi 60 MHzR-1200 NMR spectrometer or a Varian VX-300 NMR spectrometer. IR spectrawere obtained using a Midac M series FT-IR instrument. Mass spectraldata were obtained using a Finnegan MAT 90 mass spectrometer. Allmelting points are corrected.

EXAMPLE 1 5,7-Dihydroxy-4-propylcoumarin⁵ (2)

Concentrated sulfuric acid (200 mL) was added into a mixture ofphloroglucinol dihydrate (150 g, 0.926 mol) and ethyl butyrylacetate(161 g, 1.02 mol). The resulting mixture was stirred at 90° C. for twohours whereupon it was poured onto ice. The solid product was collectedby filtration, and then dissolved in ethyl acetate. The solution waswashed with brine and dried over Na₂ SO₄. After removal of the solventin vacuo, the residue was triturated with hexane to provide essentiallypure compound 2 (203 g) in quantitative yield, mp 233-235° C. (Lit.⁵236-238° C.). ¹ H-NMR⁵ (DMSO-d₆) δ0.95 (3H,t,J=6.9 Hz, CH₃); 1.63 (2H,apparent sextet, J=7.0 Hz, CH₂); 2.89 (2H,t,J=7.5Hz,CH₂); 5.85 (1H, s,H₃); 6.22 (1H, d, J=2.0 Hz, H₆); 6.31 (1H, d, J=2.0 Hz, H₈); 10.27 (1H,s, OH); 10.58 (1H, s, OH); MS (EI); 220(100, M⁺); 205 (37.9, M-CH₃); 192(65.8, M-C₂ H₄); 177 (24.8, M-C₃ H₇); 164 (60.9, M-CHCO₂ +1); 163 (59.6M-CHCO₂); IR (KBr): 3210 (vs and broad, OH); 1649 (vs, sh); 1617 (vs,sh); 1554 (s) cm⁻¹ ; Anal. calcd. for C₁₂ H₂₄ O₄ : C, 65.45; H. 5.49;Found: C, 65.61; H, 5.44.

EXAMPLE 2 5,7-Dihydroxy-8-propionyl-4-propylcoumarin (3)

A three-neck flask (500 mL) equipped with an efficient methanicalstirrer, thermometer and addition funnel was charged with5,7-dihydroxy-4-propylcoumarin, 2, (25.0 g, 0.113 mol), aluminumchloride (62.1 g; 0.466 mol), and nitrobenzene (150 mL) and the mixturewas stirred until a solution was obtained, which was cooled to 0° C. inan ice bath. A solution of propionyl chloride (15.2 g; 0.165 mol) incarbon disulfide (50 mL) was added dropwise at such a rate that thereaction temperature was maintained at 8-10° C. Addition was completedover a period of 1 hour with vigorous stirring. The reaction wasmonitored by TLC using a mobile phase of 50% ethyl acetate/hexane. Afterthree hours, an additional portion of propionyl chloride (2.10 g; 0.0227mol) in carbon disulfide (10 mL) was added. Immediately after the TLCanalysis indicated the total consumption of starting material, thereaction mixture was poured onto ice, and allowed to stand overnight.The nitrobenzene was removed by steam distillation, and the remainingsolution was extracted several times with ethyl acetate. The extractswere combined and dried over Na₂ SO₄. The crude product obtained byevaporation in vacuo was purified by chromatography on a silica gelcolumn eluting with 50% ether/hexane to provide the desiredpropionylated coumarin 3, mp (corr) 244-246° C. ¹ H-NMR (DMSO-d₆) δ0.96(3H, t, J=7.3 Hz, CH₃); 1.10 (3H, t, J=7.2 Hz, CH₃); 1.60 (2H, m, CH₂);2.88 (2H, t, J=7.7 Hz, CH₂); 3.04 (2H, q, J=7.2 Hz, CH₂); 5.95 (1H, s,H₃); 6.31 (1H, s, H₆); 11.07 (1H, s, OH); 11.50 (1H, s, OH); MS (EI):277 (6.6, M+1); 276 (9.0, M⁺); 247 (100, M-C₂ H₅); IR (KBr): 3239 (s andbroad, OH); 1693 (s, C═O), 1625 and 1593 (s) cm⁻¹ ; Anal. calcd. for C₁₅H₁₆ O₅ : C, 65.21; H, 5.84; Found: c, 64.92; H, 5.83. The isomerassignment was made by analogy to precedent.¹⁵

EXAMPLE 32,2-Dimethyl-5-hydroxy-6-propionyl-10-propyl-2H,8H-benzo[1,2-b:3,4-b']-dipyran-8-one(4)

A mixture of 3 (2.60 g, 9.42 mmol) and 4,4-dimethoxy-2-methylbutan-2-ol(5.54 g, 37.7 mmol) were dissolved in anhydrous pyridine (6.5 mL). Themixture was ref luxed under nitrogen for three days. After removal ofthe solvent in vacuo, the residue was dissolved in ethyl acetate. Theethyl acetate was washed several times with 1 N HCl and brine. It wasthen dried over Na₂ SO₄. The crude product obtained by evaporation invacuo was purified by silica gel column chromatography, eluting with 25%ethyl acetate/hexane to afford 2.55 g of 4 in 78.6% yield, mp 96-98° C.¹ H-NMR (CDCl₃) δ1.05 (3H, t, J=7.3 Hz, CH₃); 1.22 (3H, t, J=7.5 Hz,CH₃); 1.53 (6H, s, 2 CH₃); 1.75 (2H, m, CH₂); 2.92 (2H, t, J=7.1 Hz,CH₂); 3.35 (2H, q, J=7.1 Hz, CH₂); 5.56 (1H, d, J=10.0 Hz, H₃); 5.98(1H, s, H₉); 6.72 (1H, d, J=10.0 Hz, H₄); MS (EI): 343 (5.7, M+1); 342(22.5, M⁺); 327 (100, M-CH₃); IR (KBr): 1728 (vs, C═O) cm⁻¹ ; Anal.calcd. for C₂₀ H₂₂ O₅ : C, 70.16; H, 6.48; Found: C, 70.45; H, 6.92.

EXAMPLE 4 10,11-Didehydro-12-oxocalanolide A (5)

A mixture of 4 (1.76 g, 5.11 mmol) and sodium acetate (0.419 g, 5.11mmol) in acetic anhydride (12 mL) were refluxed for 10 hours whereuponthe solvent was removed in vacuo. The residue was purified by silica gelcolumn chromatography, eluting first with 25% ethyl acetate/hexanefollowed by 50% ethyl acetate/hexane to provide 1.16 g (62% yield) ofenone S(6,6,10,11-tetramethyl-4-propyl-2H,6H,12H-benzo[1,2-b:3,4-b':5,6-b"]-tripyran-2,12-dione)as a white solid, mp 209-209.5° C. ¹ H-NMR (CDCl₃) δ1.05 (3H, t, J=6.6Hz, CH₃); 1.56 (6H, s, 2 CH₃); 1.73 (2H, m, CH₂) ; 1.98 (3H, s, CH₃) ;2.38 (3H, s, CH₃); 2.91 (2H, t, J=7.5 Hz, CH₂); 5.69 (1H, d, J=10.0 Hz,H₇); 6.11 (1H, s, H₃); 6.71 (1H, d, J=10 Hz, H₈); MS (EI): 366 (29.6,M⁺); 351 (100, M-CH₃); 323 (16.5, M-C₃ H₇); IR (KBr): 1734 (vs, C═O),1657, 1640, 1610, and 1562 cm⁻¹ ; Anal. calcd. for C₂₂ H₂₂ O₅ : 72.12;H, 6.05; Found: C, 72.14; H, 6.15.

EXAMPLE 5 10,11-Didehydrocalanolide A (6)

A mixture of enone 5 (160 mg, 0.437 mmol) and tri-n-butyltin hydride(0.318 g, 1.09 mmol) in dry dioxane (2.0 mL) was refluxed under nitrogenfor 12 hours. The solvent was then removed in vacuo and the residue waspurified by preparative TLC using 25% ethyl acetate in hexane as themobile phase. The product exhibited an R_(f) of about 0.4. Enol 6(12-hydroxy-6,6,10,11-tetramethyl-4-propyl-2H,6H,12H-benzo[1,2-b:3,4-b':5,6-b"]-tripyran-2-one)(13.3 mg, 8%) was isolated as an oil from the plate by ethyl acetateelution. This elution may have been inefficient, and the actual yieldhigher, as indicated by analytical TLC of the crude product. ¹ H-NMR(CDCl₃) δ0.92 (3H, t, J=6.0 Hz, CH₃); 1.26 (3H, s, CH₃); 1.39 (3H, s,CH₃); 1.63 (2H, m, CH₂) ; 1.96 (3H, s, CH₃) ; 2.36 (3H, s, CH₃) ; 2.45(2H, t, J=6.0 Hz, CH₂); 3.65 (1H, s, H,₂); 5.51 (1H, d, J=10.0 Hz, H.);6.06 (1H, S, H₃); 6.67 (1H, d, J =10.0 Hz, H₈); 13.25 (1H, br s, OH); MS(EI): 369 (3.8, M+1), 368 (4.4, M⁺), 367 (8.3, M-1) 366 (28.4, M-2), 351(100, M-OH); IR(KBr): 1651 (s), 1589 (m)cm⁻¹.

EXAMPLE 6 12-Oxocalanolide A [(±)-(7)

A solution containing chromene 4 (344 mg, 1.0 mmol), acetaldehydediethylacetal (473 mg, 4.0 mmol), trifluoroacetic acid (1.5 mL, 19.4mmol) and anhydrous pryidine (0.7 mL) was heated at 140° C. under N₂.The reaction was monitored by TLC analysis. After 4 hours, the reactionmixture was cooled to room temperature, diluted with ethyl acetate andwashed several times with 10% aqueous NaHCO₃ and brine. The organiclayer was separated and dried over Na₂ SO₄. The solvent was removed invacuo and the crude product was purified by silica gel columnchromatography eluting with ethyl acetate/hexane (2:3). Chromanone (±)-7 (10,11-trans-dihydro-4-propyl-6,6,10,11-tetramethyl-2H,6H,12H-benzo[1,2-b:3,4-b':5,6-b"]-tripyran-2,12-dione)(110 mg, 30% yield) was obtained m.p. 176-177° C. (Lit.⁵ 130˜132° C.). ¹HNMR⁵ (CDCl₃) δ1.02 (3H, t, J=7.5 Hz, CH₃); 1.21 (3H, d, J=6.8 Hz, CH₃);1.51 (3H, d, J=7.0 Hz, CH₃); 1.55 (6H, 2s, 2 CH₃); 1.63 (2H, sextet,J=7.0 Hz, CH₂); 2.55 (1H, dq, J=6.9 Hz, J=11.0 Hz, H₁₁); 2.88 (2H, t,J=7.6 Hz, CH₂); 4.28 (1H, dq, J=6.3 Hz, J=11.0 Hz, H₁₀); 5.60 (1H, d,J=9.9 Hz, H₇); 6.04 (1H, s, H₃); 6.65 (1H, d, J=11.8 Hz, H₈); MS (CI):369 (100, M+1).

EXAMPLE 7 (±)-Calanolide A (1)

To a solution of chromanone (±)-7 (11 mg, 0.03 mmol) in ethanol (0.4 mL)was added sodium borohydride (2.26 g, 0.06 mmol) and CeCl₃ (H₂ O)₇ (11.2mg, 0.03 mmol) in ethanol (5 mL) at room temperature. After stirring for45 minutes, the mixture was diluted with H₂ O and extracted with ethylacetate. The organic layer was dried over Na₂ SO₄ and concentrated. Thecrude product was purified by preparative TLC eluting with ethylacetate/hexane (1:1) to afford (±)-calanolide A (1) (10.5 mg, 94%). m.p.52-54° C., which increased to 102° C. after it was dried thoroughly(Lit⁵. 56-58° C.). ¹ H NMR (CDCl₃): δ1.03 (3H, t, J=7.3Hz, CH₃), 1.15(3H, d, J=6.8Hz, CH₃), 1.46 (3H, d, J=6.8Hz, CH₃), 1.47 (3H, s, CH₃),1.51 (3H, s, CH₃), 1.66 (2H, m, CH₂), 1.93 (1H, m, H,,), 2.89 (2H, m,CH₂), 3.52 (1H, broad-s, OH), 3.93 (1H, m, H₁₀), 4.72 (1H, d, J=7.8Hz,H₁₂), 5.54 (1H, d, J=10.0Hz, H₇), 5.94 (1H, s, H₃), 6.62 (1H, d,J=9.9Hz, H₈); MS (CI): 371 (75.4, M+1), 370 (16.1, M+), 353 (100, M-OH);Anal. calcd. for C₂₂ H₂₅ O₅ : C, 71.33; H, 7.07; Found: C, 71.63; H,7.21.

EXAMPLE 8 5,7-Dihydroxy-4-propylcoumarin (2)

In this Example, kilogram scale preparation of intermediate 2 isdescribed. Into a stirring suspension of phloroglucinol (3574.8 g, 28.4mol, pre-dried to constant weight) and ethyl butyrylacetate (4600 mL,28.4 mol) was added concentrated sulfuric acid dropwise at such a ratethat the internal temperature did not exceed 40° C. After 100 mL ofsulfuric acid was added, the temperature rose to 70° C. and thesuspension turned into a yellow solid. Analysis of TLC indicated thatthe reaction had proceeded to completion. The reaction mixture wasdiluted with water (10 L) and stirred at ambient temperature overnight.The precipitated product was collected by filtration and then rinsedwith water until the filtrate was neutral. A quantity of 4820 g (77%yield) of 5,7-dihydroxy-4-propylcoumarin 2 was obtained after beingdried, which was identical with an authentic sample by comparsion ofTLC, melting point and spectroscopic data.

EXAMPLE 9 5,7-Dihydroxy-8-propionyl-4-propylcoumarin (3)

In this Example, kilogram quantities of intermediate 3 were synthesizedusing propionic anhydride instead of propionyl chloride.5,7-dihydroxy-4-propylcoumarin 2 (1710 g, 7.77 mol) and AlCl₃ (1000 g,7.77 mol) were mixed in 1,2-dichloroethane (9 L). The resulting orangesuspension was stirred and heated to 70° C. until a solution wasobtained. Then, a mixture of propionic anhydride (1010 g. 7.77 mol) andAlCl₃ (2000 g, 15.54 mol) in 1,2-dichloroethane (3.4 L) was addeddropwise over 3 h. The reaction was allowed to stir at 70° C. for anadditonal hour. After being cooled down to room temperature, thereaction mixture was poured into a rapidly stirring mixture of ice waterand IN HCl. The precipitated product was taken into ethyl acetate (30 L)and the aqueous solution was extracted with the same solvent (10 L×2).The combined extracts were successively washed with 1 N HCl (10 L),saturated aq. NaHCO₃ (10 L), and water (10 L). After being dried overMgSO₄ and concentrated in vacuo, a solid product (1765 g) was obtainedwhich was washed with ethyl acetate (15 L) and recrystallized fromdioxane (9.5 L) to provide 514 g of pure compound 3. From the ethylacetate washings, an additional 100 g of compound was obtained afterrecrystallization from dioxane. Thus, the combined yield for compound 3,which was identical with an authentic sample by comparison of TLC,melting point and spectroscopic data, was 29%.

EXAMPLE 102,2-Dimethyl-5-hydroxy-6-propionyl-10-propyl-2H,8H-benzo[1,2-b:3,4-b']dipyran-8-one(4)

In this Example, intermediate 4 was prepared in half kilogram quantitiesfrom 3 via modification of the reaction conditions described in Example3. A mixture of compound 3 (510.6 g, 1.85 mol) and4,4-dimethoxy-2-methylbutan-2-ol (305.6 g, 2.06 mol) were dissolved in amixture of toluene (1.5 L) and dry pyridine (51 mL). This mixture wasstirred and refluxed; water and methanol formed during the reaction wereremoved azeotropically via a Dean-Stark trap. The reaction was monitoredby TLC. After 6 days, the reaction had proceeded to completion. Themixture was then cooled to ambient temperature and diluted with ethylacetate (2 L) and 1 N HCl (1 L). The ethyl acetate solution wasseparated and washed with 1N HCl (500 mL) and brine (1 L). After beingdried over Na₂ SO₄ and evaporated in vacuo, a quantity of 590 g (93%yield) of compound 4 was obtained which was greater than 95% purewithout further purification and was compared with an authentic sampleby TLC and spectroscopic data.

EXAMPLE 11 12-Oxocalanolide A (7)

In this Example, chromanone (±)-7 was prepared from two alternativepathways involving either a one-step paraldehyde reaction (procedure A)or a two-step reaction process (procedures B and C).

Procedure A

Paraldehyde One-Step Reaction: To a stirring solution of chromene 4 (350mg, 1.0 mmol) and PPTS (250 mg, 1.0 mmol) in 1,2-dichloroethane (2 mL)at ambient temperature under N₂ was added 3 mL paraldehyde (22.5 mmol).The resulting mixture was refluxed for 7 h. Then, CF₃ CO₂ H (1 mL), anadditional equivalent of PPTS and 1 mL of paraldehyde were added; themixture was refluxed overnight. The reaction mixture was neutralizedwith saturated aqueous NaHCO₃ and extracted with ethyl acetate (50mL×3). The crude product obtained by evaporation under reduced pressurewas washed with hexane. The residue was purified by columnchromatography eluting with ethyl acetate/hexane (1:2) to afford 100 mg(27% yield) of chromanone (±)-7 and 30 mg (8% yield) of (±)-7a.Chromanone (±)-7(10,11-trans-dihydro-4-propyl-6,6,10,11-tetramethyl-2H,6H,12H-benzo[1,2-b:3,4-b':5,6-b"]tripyran-2,12-dione)obtained by this method was identical with an authentic sample bycomparison of TLC, HPLC and spectroscopic data.

Procedure B

LDA/Sulfuric Acid Two-Step Reaction: To a stirring solution of chromene4 (5.0 g, 14.6 mmol) in THF (75 mL) at -30° C. under N₂ was added 18.3mL (36.5 mmol) of 2 M LDA in THF. After 15 min at the same temperature,acetaldehyde (5.0 mL, 89.5 mmol) was added via syringe. The reaction wasmonitored by TLC analysis. After 1 h, the reaction mixture was quenchedat -10° C. with saturated aqueous NH₄ Cl (75 mL) and extracted withethyl acetate (125 mL×3). The combined extracts were washed with brine(125 mL) and dried over Na₂ SO₄. Removal of solvents in vacuo afforded areddish oil of (±)-8a and (±)-8b (8.5 g).

The crude (±)-8a and (±)-8b was dissolved in acetic acid (100 mL) andthen 50% H₂ SO₄ (100 mL) was added with stirring. The resulting mixturewas heated at 75° C. for 2.5 h and then at 50° C. for 4 h. TLC analysisindicated that the starting material had been consumed. The reactionmixture was determined to contain both chromanone (±)-7 and10,11-cis-dimethyl derivative (±)-7a in a 1:1 ratio. After cooling toambient temperature, the reaction mixture was poured into a mixture ofice water (500 mL) and ethyl acetate (500 mL). The layers were separatedand the aqueous layer was extracted with ethyl acetate (200 mL ×3). Theethyl acetate solutions were combined and washed with saturated aqueousNaHCO₃ and brine. After being concentrated in vacuo, the product waspurified by chromatography on a silica gel column eluting with ethylacetate/hexane (2:3) to provide 850 mg (16% yield) of chromanone (±)-7,which was further purified by recrystallation from ethyl acetate/hexaneand was identical with an authentic sample by comparison of TLC, HPLCand spectroscopic data.

Procedure C

LDA/Mitsunobu Two-Step Reaction: Into a stirring solution of THF (10 mL)containing triphenylphosphine (1.27 g, 4.80 mmol) and the crude mixtureof (±)-8a and (±)-8b, obtained from chromene 4 (1.0 g, 2.34 mmol), 2.5equivalents of LDA and 6.0 equivalents of acetaldehyde by the proceduredescribed above, was added dropwise diethyl azodicarboxylate (DEAD, 0.77mL, 4,89 mmol). The resulting reddish solution was stirred at ambienttemperature under N₂ for 1 h, after which the reaction mixture wasquenched with saturated aqueous NH₄ Cl and extracted with ethyl acetate(50 mL×3). The extracts were washed with brine and dried over Na₂ SO₄.After removal of solvents, the crude product was purified by columnchromatography on silica gel eluting with ethyl acetate/hexane (2:3) toprovide 412 mg (48% yield, based on chromene 4) of chromanone (±)-7, thepredominant product of the reaction, which was identical with anauthentic sample by comparison of TLC, HPLC and spectroscopic data.

EXAMPLE 12 (±)-Calanolide A (1)

In this Example, (±)-calanolide A was prepared in multi-gram scale usingthe procedure described in Example 7. To a stirring solution ofchromanone (±)-7 (51.5 g, 0.14 mol) in ethanol (1.5 L) was added CeCl₃(H₂ O)₇ (102 g, 274 mmol). The mixture was stirred for 1.5 h at roomtemperature under N₂ and then cooled to -30° C. with an ethyleneglycol/H₂ O (1:2 w/w) dry ice bath. After the temperature wasequilibrated to -30° C., NaBH₄ (21.3 g, 563 mmol) was added and stirredat the same temperature for 8.5 h, at which time the reaction wasquenched with H₂ O (2 L) and extracted with ethyl acetate (2 L×3). Theextracts were combined, washed with brine (2 L) and dried over Na₂ SO₄.The crude product obtained by removal of solvent under reduced pressurewas passed through a short silica gel column to provide 53 g of mixturewhich contained 68% of (±)-calanolide A, 14% of calanolide B and 13% ofchromanone (±)-7 as shown by HPLC. This material was subjected tofurther purification by preparative HPLC to afford pure (±)-calanolide A(1).

EXAMPLE 13 Chromatographic Resolution of Synthetic (±)-Calanolide A

The synthetic(±)-1 was resolved into enantiomers, (+)-calanolide A and(-)-calanolide A, by preparative HPLC¹⁶. Thus, using a normal phasesilica gel HPLC column (250 mm×4.6 mm I. D. Zorbasil, 5 μm particlesize, MAC-MOD Analytical, Inc., PA, USA), the synthetic (±)-1 appearedas one peak with a retention time of 10.15 minutes when hexane/ethylacetate (70:30) was used as the mobile phase at a flow rate of 1.5mL/min and a wavelength of 290 nm was used as the uv detector setting.However, on a chiral HPLC column packed with amylose carbamate (250mm×4.6 mm I.D. Chiralpak AD, 10 μm particle size, Chiral Technologies,Inc., PA, USA), two peaks with retention times of 6.39 and 7.15 minutesin a ratio of 1:1 were observed at a flow rate of 1.5 mL/min. The mobilephase was hexane/ethanol (95:5) and the uv detector was set at awavelength of 254 nm. These two components were separated using asemi-preparative chiral HPLC column, providing the pure enantiomers ofcalanolide A. The chemical structures of the separated enantiomers,which were assigned based on their optical rotations and compared withthe reported natural product, were characterized by spectroscopic data.HPLC chromatograms of (±)-calanolide A and its optical forms are shownin FIG. 6.

(+)-Calanolide A (1)

mp 47-50° C. (Lit.¹⁷ 45-48° C.); [α]²⁵ _(D) =+68.8°(CHCl₃, c 0.7)(Lit.¹⁷ [a]²⁵ _(D) =+66.6°) (CHCl₃ ; c 0.5); ¹ H NMR (CDCl₃) δ1.03 (3H,t, J=7.3 Hz, CH₃), 1.15 (3H, d, J=6.8 Hz, CH₃), 1.46 (3H, d, J=6.4 Hz,CH₃), 1.47 (3H, s, CH₃), 1.51 (3H, s, CH₃), 1.66 (2H, m, CH₂), 1.93 (1H,m, H₁₁), 2.89 (2H, m, CH₂), 3.52 (1H, d, J=2.9 Hz, OH), 3.93 (1H, m,H₁₀), 4.72 (1H, dd, J=7.8 Hz, J=2.7 Hz, H₁₂), 5.54 (1H, d, J=9.9 Hz,H₇), 5.94 (1H, s, H₃), 6.62 (1H, d, J=9.9 Hz, H₈); ¹³ C NMR (CDCl₃) S13.99 (CH₃), 15.10 (CH₃), 18.93 (CH₃), 23.26 (CH₂), 27.38 (CH₃), 28.02(CH₃), 38.66 (CH₂), 40.42 (CH), 67.19 (CH--OH), 77.15 (CH--O), 77.67(C--O), 104.04 (C_(4a)), 106.36 (C_(8a) and C_(12a)), 110.14 (C₃),116.51 (C₈), 126.97 (C₇) , 151.14 (C_(4b)) 153. 10 (C_(8b)), 154.50(C_(12b)), 158. 88 (C₄), 160.42 (C═O); CIMS: 371 (100, M+1), 370(23.6,M⁺), 353 (66.2, M--OH); IR: 3611 (w) and 3426 (m, broad, OH), 1734(vs. C═O), 1643 (m), 1606 (m) and 1587 (vs) cm⁻¹ ; UV λ_(max)(methanol): 204 (32,100), 228 (23,200), 283 (22,200), 325 (12,700) nm;Anal. calcd. for C22H₂₆ O₅ 1/4H₂ O: C, 70.47; H, 7.12; Found: C, 70.64;H, 7.12.

(-)-CalanolideA (1)

mp 47-50° C.;[α]²⁵ _(D) =-75.6°(CHCl₃, c 0.7) Lit.¹⁷ [α]²⁵ _(D) =-66.6°(CHCl₃, c 0.5); ¹ H NMR (CDCl₃) δ1.03 (3H, t, J=7.4 Hz, CH₃), 1.15 (3H,d, J=6.8 Hz, CH₃), 1.46 (3H, d, J=6.3 Hz, CH₃), 1.47 (3H, s, CH₃), 1.51(3H, s, CH₃), 1.66 (2H, m, CH₂), 1.93 (1H, m, H₁₁), 2.89 (2H, m, CH₂),3.50 (1H, d, J=2.9 Hz, OH), 3.92 (1H, m, H₁₀), 4.72 (1H, dd, J=7.8 Hz,J=2.7 Hz, H₁₂), 5.54 (1H, d, J=10.0 Hz, H₇), 5.94 (1H, s, H₃), 6.62 (1H,d, J=10.0 Hz, H₈); ¹³ C NMR (CDCl₃) δ13.99 (CH₃), 15.10 (CH₃), 18.93(CH₃), 23.36 (CH₂), 27.38 (CH₃), 28.02 (CH₃), 38.66 (CH₂), 40.42 (CH),67.19 (CH--OH), 77.15 (CH--O), 77.67 (C--O), 104.04 (C_(4a)), 106.36(C_(8a) and C_(12a)), 110.14 (C₃), 116.51 (C₈), 126.97 (C₇), 151.14(C_(4b)) , 153.11 (C_(8b)) , 154.50 (C₁₂ b) , 158.90 (C₄), 160.44 (C═O); CIMS: 371 (95.2, M+1), 370 (41.8,M⁺), 353 (100, M--OH); IR: 3443 (m,broad, OH), 1732 (vs, C═O) , 1643 (m) , 1606 (m) and 1584 (vs) cm⁻¹ ; UVλ_(max) (methanol): 200 (20,500), 230 (19,400), 283 (22,500), 326(12,500) nm; Anal. calcd. for (C₂₂ H₂₆ O₅ 1/4H₂ O: C, 70.47; H, 7.12;Found: C, 70.27; H, 7.21.

EXAMPLE 14 Enzymatic Resolution of (±)-Calanolide A

To a magnetically stirred suspension of (±)-calanolide A, prepared bythe method of the present invention, and vinyl butyrate (0.1 mL) inhexane (0.5 mL) at ambient temperature was added 1 mg of lipase PS-13(Pseudomonas Species) (Sigma Corporations, St. Louis, Mo., USA). Thereaction mixture was stirred and monitored by conventional means such asTLC analysis. At 10 days, an additional 1 mg of lipase PS-13 was added.After stirring for a total of 20 days, the reaction was stopped becausethere was no obvious increase in ester formation. The enzyme wasfiltered out and the filtrate was concentrated to dryness. The residuewas analyzed by HPLC (see Example 13), which showed that 21% of(-)-calanolide A had been converted into its butyrate ester form. Theenriched (+)-calanolide A and the butyrate ester of (-)-calanolide A canbe easily separated by conventional means such as column chromatography.The enriched (+)-calanolide A may be repeatedly treated with vinylbutyrate and lipase PS-13 as described above so as to obtain high e.e.of (+)-calanolide A.

EXAMPLE 15 Aldol Reaction (Scheme III) of Chromene 4 in the Presence ofLDA

To a stirring solution of chromene 4 (1.0 g, 2.9 mmol) in THF (15 mL) at-78° C. under N₂ was added 2 M LDA in THF (3.2 mL, 6.4 mmol). After 1 hat the same temperature, acetaldehyde (1.0 mL, 17.5 mmol) was added viasyringe. The reaction was monitored by TLC analysis. After 1 h, thereaction mixture was quenched with a precooled 2 N HCl in methanol (15mL) and extracted with ethyl acetate (30 mL×3). The combined extractswere washed with brine and dried over Na₂ SO₄. Removal of solvents invacuo afforded a reddish oil, which was purified by silica gel columnchromatography eluting with a discontinuous gradient of 5%, 10%, 15%,25% and 30% of ethyl acetate in hexane to obtain 780 mg (70% yield) of amixture of (±)-8a and (±)-8b in a ratio of 1:1, as indicated by ¹ H NMR.Pure samples of (±)-8a and (±)-8b were obtained by carefully collectingthe front fractions and later fractions from column chromatography,analytical data of which were described below:

6,6-Dimethyl-9-hydroxy-10-[2(S*)-methyl-3(R*)-hydroxybutyro]-4-propyl-2H,6H-benzo[1, 2-b: 3, 4-b']dipyran-2-one [syn-(±)8a]

m.p. 66-67° C.; ¹ H NMR (CDCl₃): 1.05 (3H, t, J=7.3 Hz, CH₃), 1.30 (3H,d, J=6.0 Hz, CH₃), 1.33 (3H, d, J=6.6 Hz, CH₃), 1.54 (6H, s, 2 CH₃),1.67 (2H, m, CH₂), 2.62 (1H, broad-s, OH), 2.91 (2H, t, J=7.7 Hz, CH₂),3.98 (1H, dq, J=2.7 Hz, J=7.0 Hz, H₂), 4.29 (1H, m, H₃), 5.59 (1H, d,J=10.0 Hz, H₇), 6.01 (1H, s, H₃), 6.73 (1H, d, J=10.0 Hz, H₈), 14.11(1H, s, OH); 1H NMR (DMSO-d₆): 1.00 (3H, t, J=7.3 Hz, CH₃), 1.13 (3H, d,J-6.6 Hz, CH₃), 1.16 (3H, d, J=6.8 Hz, CH₃), 1.49 (3H, s, CH₃), 1.50(3H, s, CH₃), 1.60 (2H, apparent sextet, J=7.6 Hz, CH₂), 2.88 (2H,apparent dd, J=6.3 Hz, J=9.0 Hz, CH₂), 3.39 (1H, broad-s, OH), 3.68 (1H,dq, J=5.2 Hz, J=6.7 Hz, H₂), 3.97 (1H, apparent quintet, J=5.8 Hz, H₃),5.78 (1H, d, J=10.1 Hz, H₇), 6.11 (1H, s, H₃), 6.63 (1H, d, J=10.1 Hz,H₈), 13.25 (1H, s, OH); MS (CI): 388 (36.5, M+2), 387 (100, M+1), 386(6.6, M⁺), 369 (21.6, M--OH), 343 (50.7, M--C₃ H₇); UV (methanol) nm:199 (41,000), 270 (25,700), 306 (21,900); IR (KBr) cm⁻¹ : 3395 (broad,m, OH), 1734 (s) and 1707 (vs) (C═O), 1644 (m), 1608 (vs), 1578 (vs) and1547 (vs); Anal. Calcd. for C₂₂ H₂₆ O₆.1/3H₂ O: C, 67.33; H, 6.84;Found: C, 67.43; H, 6.93.

6,6-Dimethyl-9-hydroxy-10-[2(S*)-methyl-3(S*)-hydroxybutyro]-4-propyl-2H,6H-benzo[1,2-b:3,4-b']dipyran-2-one[anti-(±)-8b]

m.p. 115° C.; ¹ H NMR (CDCl₃): 1.05 (3H, t, J=7.4 Hz, CH₃), 1.25 (3H, d,J=6.4 Hz, CH₃)₁ 1.29 (3H, d, J=6.9 Hz, CH₃), 1.54 (6H, S, 2 CH₃), 1.66(2H, apparent sextet, J=7.6 Hz, CH₂), 2.92 (2H, t, J=7.8 Hz, CH₂), 2.95(1H, d, J=5.5 Hz, OH), 3.98 (1H, dq, J=6.1 Hz, J=6.8 Hz, H₂), 4.22 (1H,apparent sextet, J=6.2 Hz, H₃), 5.59 (1H, d, J=10.1 Hz, H₇), 6.03 (1H,S, H₃), 6.73 (1H, d, J=10.1 Hz, H₈), 14.25 (1H, s, OH); ¹ H NMR(DMSO-d₆): 1.00 (3H, t, J=7.3 Hz, CH₃), 1.11 (6H, d, J=6.7 Hz, 2 CH₃),1.49 (3H, s, CH₃), 1.50 (3H, S, CH₃), 1.60 (2H, apparent sextet, J=7.3Hz, CH₂), 2.85, 2.90 (2H, t-AB type, J=7.7 Hz, J_(AB) =21.4 Hz, CH₂),3.59 (1H, apparent quintet, J=7.1 Hz, H₂), 3.96 (1H, apparent quintet,J=7.0 Hz, H₃), 4.97 (1H, broad-s, OH), 5.78 (1H, d, J=10.1 Hz, H₇), 6.10(1H, S, H₃), 6.63 (1H, d, J=10.0 Hz, H₈), 12.69 (1H, S, OH); MS (EI):387 (2.8, M+1), 386 (9.4, M⁺), 371 (5.3, M--CH₃), 369 (1.5, M--OH), 353(54.0, M--CH₃ --H₂ O), 342 (22.5, M--C₃ H₇ -1), 327 (100, M--C₃ H₇--OH+1); UV (methanol) nm: 199 (41,000), 270 (25,700), 306 (21,900); IR(KBr) cm⁻¹ : 3478 (broad, m, OH), 1736 (vs) and 1707 (vs) (C═O), 1645(m), 1603 (vs), 1584 (vs, sh); Anal. Calcd. for C₂₂ H₂₆ O₆.1/3H₂ O: C,67.33; H, 6.84; Found: C, 67.34; H, 6.45.

EXAMPLE 16 Aldol Reaction (Scheme III) of Chromene 4 in the Presence ofLDA/TiCl₄

In this Example, two procedures are provided for effecting the Aldolreaction. Procedure B was found to be more suitable for scale-up becauseof simplification of temperature control.

Procedure A

To a stirring solution of chromene 4 (200 mg, 0.58 mmol) in drymethylene chloride (10 mL) at -78° C. under N₂ was added 2 M solution ofLDA in heptane/THF/ethyl benzene (0.64 mL, 1.28 mmol). The reactionmixture was stirred at -78° C. for 30 min and then TiCl₄ (0.13 mL, 1.17mmol) was added. The resulting yellow solution was warmed to -40° C. andstirred for 45 min. The mixture was recooled to -78° C., andacetaldehyde (150 mg, 3.5 mmol) was added via syringe. After 4 h, thereaction was quenched by slow addition of pre-cooled saturated NH₄ Cl(10 mL). Water (3 mL) was added to dissolve the oily solid. The mixturewas extracted with ethyl acetate (50 mL×3). The combined extracts werewashed with brine (100 mL) and dried over MgSO₄. The crude productobtained by evaporation was purified by silica gel columnchromatography, eluting with hexane/ethyl acetate (5:1) to affordunreacted chromene 4 (30 mg, 15% yield) and syn-(±)-8a (140 mg, 61%yield), which contained 7% of anti-(±)-8b as shown by HPLC.

Procedure B

To a stirring solution of chromene 4 (20 g, 58.4 mmol) in dry methylenechloride (300 mL) at -40° C. under N₂ was added TiCl₄ (19 mL, 175 mmol).The mixture was then cooled to -78° C., followed by slow addition of 2 Msolution of LDA in heptane/THF/ethyl benzene (64 mL, 128 mmol). After 30min at the same temperature, acetaldehyde (9 mL, 175 mmol) was added viasyringe. The reaction mixture was stirred at -78° C. for 2 h. TLCanalysis (hexane/ethyl acetate, 5:1) indicated that approximately 90%chromene 4 had been converted. The mixture was then poured intopre-cooled saturated NH₄ Cl (240 mL). Water (120 mL) was added todissolve the oily solid and the mixture was stirred for 20 min. Layerswere separated and the aqueous solution was extracted with ethyl acetate(600 mL×3). The combined extracts were washed with brine (600 mL) anddried over MgSO₄. Removal of solvents in vacuo afforded a reddish oil(23 g), which was taken up into ether (250 mL). The undissolved residuewas filtered and the etheral solution was concentrated to half volumeand then slowly added into rapidly stirring hexane cooled at -78° C.Precipitates thus formed were collected by filtration to afford syn-(±)-8a (11.1 g, 49% yield), which contained 4% of (±)-8b as shown by HPLC.

EXAMPLE 17 Enzymatic Resolution of syn-(±)-8a (Scheme III)

Into a stirring solution of syn-(±)-8a (7.6 g, 19.7 mmol) in tert-butylmethyl ether (130 mL) at ambient temperature under N₂ were addedsuccessively vinyl acetate (33 mL), 4 Å molecular sieves (17 g) andLipase PS-30 (3.8 g) (Amano Enzyme U.S.A. Co., Ltd., Troy, Va.). Theresulting mixture was vigorously stirred at ambient temperature for 4days, whereupon it was filtered through celite and the celite was washedwith ethyl acetate (20 mL). The crude product obtained from evaporationwas subjected to silica gel column chromatography eluting with adiscontinuous gradient of 5%, 10%, 15%, 25%, 30% and 40% of ethylacetate in hexane to afford 4.8 g (63% yield) of the acetate (9), whichwas contaminated by over-acylation product of (±)-8a, and 2.8 g (37%yield) of pure syn-(+)-8a.

6,6-Dimethyl-9-hydroxy-10-[2(R)-methyl-3(S)-hydroxybutyro]-4-propyl-2H,6H-benzo[1,2-b:3,4-b']dipyran-2-one [syn-(+)-8a]

m.p. 82-85° C.; [α]²⁵ _(D) =0° (CHCl₃, c 0.7; [α]²⁵ _(D) =0°(CHCl₃, c0.35); ¹ H NMR (CDCl₃): 1.05 (3H, t, J=7.4 Hz, CH₃), 1.31 (3H, d, J=5.6HZ, CH₃), 1.33 (3H, d, J=6.9 Hz, CH₃), 1.54 (6H, s, 2 CH₃), 1.67 (2H,apparent sextet, J=7.6 Hz, CH₂), 2.75 (1H, broad-s, OH), 2.91 (2H, t,J=7.8 Hz, CH₂), 3.98 (1H, dq, J=2.7 Hz, J=7.0 Hz, H₂), 4.30 (1H, dq,J=2.7 Hz, J=6.5 Hz, H₃,), 5.59 (1H, d, J=10.2 Hz, H₇), 6.01 (1H, s, H₃),6.72 (1H, d, J=10.3 Hz, H₈), 14.10 (1H, s, OH); ¹³ C NMR (CDCl₃); δ10.42(CH₃), 14.00 (CH₃), 20.61 (CH₃), 23.32 (CH₂), 28.31 (2 CH₃), 39.05(CH₂), 50.93 (CHCO), 68.03 (CH--O), 79.92 (C--O), 102.95 (C_(8a)),103.69 (C_(4a)), 106.12 (C₁₀), 110.60 (C₃), 115.80 (C₈), 126.51 (C₇),157.03 and 157.11 (C₉ and C_(10a)) , 158.58 (C_(4b)), 159.01 (C₄),163.13 (CO₂), 210.61 (C═O); MS (CI): 388 (33.4, M+2), 387 (100, M+1),386 (8.5, M⁺), 369 (36.3, M--OH), 343 (97.2, M--C₃ H₇); Anal . calcd.for C₂₂ H₂₆ O₆ : C, 68.38; H, 6.78; Found: C, 68.02; H, 6.62.

EXAMPLE 1810(R),11(R)-trans-Dihydro-6,6,10,11-tetramethyl-4-propyl-2H,6H,12H-benzo-[1,2-b:3,4-b':5,6-b"]tripyran-2,12-dione[Scheme III, (+)-7]

Into a stirring solution of syn-(+)-8a (2.0 g, 5.2 mmol) in THF (50 mL)were added triphenylphosphine (1.9 g, 7.2 mmol) and diethylazodicarboxylate (DEAD, 1.2 mL, 7.6 mmol). The resulting reddishsolution was stirred at ambient temperature under N₂ for 5 h, afterwhich the reaction mixture was quenched with saturated aqueous NH₄ Cl(20 mL) and extracted with ethyl acetate (50 mL×3). The combinedextracts were washed with brine (50 mL) and dried over Na₂ SO₄. Thecrude product (5.8 g) obtained by evaporation was purified by columnchromatography on silica gel eluting with a discontinuous gradient of10%, 20%, 30% and 40% of ethyl acetate in hexane to afford 1.2 g (63%yield) of pure (+)-7. mp 171-175° C.; [α]²⁵ _(D) =+37.9°(CHCl₃, c 0.73);¹ H NMR [CDCl₃ /CD₃ OD (3:1) ]: 1.06 (3H, t, J=7.3 Hz, CH₃), 1.22 (3H,d, J=7.0 Hz, CH₃), 1.54 (3H, s, CH₃), 1.57 (3H, d, J=6.0 Hz, CH₃), 1.58(3H, s, CH₃), 1.67 (2H, apparent sextet, J=7.6 Hz, CH₂), 2.59 (1H, dq,J=6.9 Hz, J=11.1 Hz, H₁₁), 2.92 (2H, t, J=7.8 Hz, CH₂), 4.37 (1H, dq,J=6.3 Hz, J=11.1 Hz, H₁₀), 5.66 (1H, d, J=10.1 Hz, H₇), 6.05 (1H, s,H₃), 6.67 (1H, d, J=10.1 Hz, H₈); ¹³ C NMR [CDCl₃ /CD₃ OD (3:1)]: δ 9.87(CH₃), 13.34 (CH₃), 18.97 (CH₃), 22.85 (CH₂), 27.40 and 27.73 (2 CH₃),38.38 (CH₂), 46.82 (CHCO), 79.17 (CH-O and C-O), 102.91 (C_(8a)), 104.11(C_(4a)), 105.46 (C_(12a)), 111.09 (C₃), 115.21 (C₈), 126.90 (C₇),154.83 and 155.86 (C_(8a) and C_(12b)), 157.89 (C_(4b)), 158.99 (C₄),160.27 (CO₂), 190.50 (C═O); MS (CI): 370 (49.0, M+2), 369 (100, M+1),368 (17.2, M⁺); Anal. Calcd. for C₂₂ H₂₄ O₅ : C, 71.72; H, 6.57; Found:C, 71.46; H, 6.60.

(+)-Calanolide A

To a stirring solution of (+)-7 (660 mg, 1.79 mmol) in ethanol (18 mL)were added CeCl₃ (H₂ O)₇ (2.7 g, 7.17 mmol) and triphenylphosphine oxide(2.0 g, 7.17 mmol). The mixture was stirred for 1 h at ambienttemperature under N₂ and then cooled to -30° C. with an ethyleneglycol/H₂ O (1:2 w/w) dry ice bath. After the temperature wasequilibrated to -30° C., NaBH₄ (271 mg, 7.17 mmol) was added and stirredat the same temperature for 5.5 h, at which time the reaction wasquenched with saturated NH₄ Cl (20 mL) and extracted with ethyl acetate(30 mL ×3). The combined extracts were washed with brine (50 mL) anddried over Na₂ SO₄. The crude product obtained by removal of solventunder reduced pressure was purified by column chromatography on silicagel eluting with 20% of ethyl acetate in hexane to afford 520 mg (78%yield) of a mixture containing 90% of (+)-calanolide A [(+)-1] and 10%of (+)-calanolide B. (+)-Calanolide A [(+)-1] was further separated from(+)-calanolide B by normal phase HPLC and was identical with anauthentic sample.

EXAMPLE 19 Enzymatic Resolution (Scheme IV) of anti-(±)-8b

Into a stirring solution of anti-(±)-8b (3.0 g, 7.8 mmol) in tert-butylmethyl ether (78 mL) at ambient temperature under N₂ were addedsuccessively vinyl acetate (26 mL), 4 Å molecular sieves (3.0 g) andLipase PS-30 (1.5 g) (Amano Enzyme U.S.A. Co., Ltd., Troy, Va.). Theresulting mixture was vigorously stirred at ambient temperature for 41h, whereupon it was filtered through the celite and the celite waswashed with ethyl acetate (20 mL). The crude yellowish solid product(3.2 g) obtained from evaporation was purified by silica gel columnchromatography eluting with a discontinuous gradient of 5%, 10%, 15%,25%, 30% and 40% of ethyl acetate in hexane to afford 1.68 g (50% yield)of the acetate (10) and 1.37 g (46% yield) of anti-(+)-8b.

6,6-Dimethyl-9-hydroxy-10-[2(S)-methyl-3(S)-hydroxybutyro]-4-propyl-2H,6H-benzo-[1,2-b:3,4-b']dipyran-2-one[anti-(+)-8b]

m.p. 131-134° C.; [a]²⁵ _(D) =+45.3° (CHCl₃, c 0.72); ¹ H NMR (CDCl₃):1.06 (3H, t, J=7.3 Hz, CH₃), 1.25 (3H, d, J=6.6 Hz, CH₃), 1.29 (3H, d,J=6.7 Hz, CH₃), 1.55 (6H, s, 2 CH₃), 1.67 (2H, apparent sextet, J=7.6Hz, CH₂), 2.92 (2H, t, J=7.8 Hz, CH₂), 2.96 (1H, d, J=7.1 Hz, OH), 3.98(1H, apparent quintet, J=6.1 Hz, H₂), 4.22 (1H, apparent sextet, J=6.0Hz, H₃), 5.60 (1H, d, J=10.1 Hz, H₇), 6.03 (1H, s, H₃), 6.73 (1H, d,J=10.1 Hz, H₈), 14.25 (1H, s, OH); MS (CI): 388 (41.4, M+2), 387 (100,M+1), 386 (13.0, M⁺), 369 (42.8, M-OH), 343 (63.8, M-C₃ H₇); Anal.calcd. for C₂₂ H₂₆ O₆ : C, 68.38; H, 6.78; Found: C, 68.50; H, 6.91.

6,6-Dimethyl-9-hydroxy-10-[2(R)-mothyl-3(R)-acetoxybutyro]-4-propyl-2H,6H-benzo-[1,2-b:3,4-b']dipyran-2-one[anti-(+)-10]

m.p. 61-64° C.; [a]²⁵ _(D) =+30.0° (CHCl₃, c 0.73); ¹ H NMR (CDCl₃):1.06 (3H, t, J=7.2 Hz, CH₃), 1.29 (3H, d, J=6.2 Hz, CH₃), 1.32 (3H, d,J=6.7 Hz, CH₃), 1.54 (6H, s, 2 CH₃), 1.67 (2H, apparent sextet, J=7.6Hz, CH₂), 1.93 (3H, s, CH₃ CO), 2.91 (2H, m, CH₂), 4.18 (1H, dq, J=8.3Hz, J=6.9 Hz, H₂), 5.34 (1H, dq, J=8.2 Hz, J=6.4 Hz, H₃), 5.59 (1H, d,J=10.1 Hz, H₇), 6.02 (1H, s, H₃), 6.73 (1H, d, J=10.1 Hz, H₈), 14.02(1H, s, OH); MS (CI): 430 (37.1, M+2), 429 (95.2, M+1), 428 (7.2, M⁺),369 (100, M-AcO); Anal. calcd. for C₂₄ H₂₈ O₇ : C, 67.28; H, 6.59;Found: C, 67.75: H, 6.90.

EXAMPLE 20 5,7-Dihydroxy-4-trifluoromethylcoumarin (Scheme V, 11a, R₁=CF₃, R₂ =H)

Into a mixture of anhydrous phloroglucinol (8 g, 63.0 mmol) and ethyl4,4,4-trifluoroacetoacetate (12 g, 65.0 mmol) was added concentrated H₂SO₄ (11 mL). The resulting mixture was heated at 100° C. and stirred for2h, whereupon the reaction mixture was cooled to room temperature. Ice(100 g) and H₂ O (150 mL) were then added while cooling with ice bath.The precipitated product was collected and dissolved in AcOEt (100 mL),which was washed with H₂ O and dried over Na₂ SO₄. The crude product (16g) obtained by evaporation under vacuum was chromatographed in methylenechloride-ethanol (95:5) to furnish 11a (6 g, 39% yield) along withanother unidentified product. 11a: m.p. 250-252° C. afterrecrystallization from methylene chloride-hexane. ¹ H NMR (DMSO-d₆):6.30 (1H, s, H₃), 6.33 and 6.54 (2H, 2 s, H₇ and H₈), 10.68 and 10.99(2H, 2 s, 2 OH); MS (CI) m/z: 246 (100, M⁺), 226 (14.6, M-HF), 218(10.0, M-CO), 198 (59.6, M-HF-CO); IR (KBr) cm⁻¹ : 3537(m, sh) and 3384(s, broad, OH), 1709 (s, C═O), 1618 (s, C═C--C═O), 1154 (s, C-F); Anal.Calcd. for C₁₀ H₅ F₃ O₄ : C, 48.80; H, 2.05; Found, C, 48.83; H, 2.10.

EXAMPLE 21 5,7-Dihydroxy-8-isobutyryl-4-propylcoumarin (Scheme V, 12a,R₁ =n-Pr, R₂ =H, R₃ =R₄ =Me)

Into a flame-dried 500 mL 3-necked round-bottom flask was placed5,7-dihydroxy-4-propylcoumarin (2, 10.0 g, 48.1 mmol) and AlCl₃ (12.0 g,90 mmol) under N₂. Dichloroethane (120 mL) was then added, and thesolution warmed to 75° C. with a water bath with mechanical stirring.After stirring 15 min at 75° C., a homogenous solution was obtained. Tothis solution was added a mixture of isobutyric anhydride (7.61 g, 48.1mmol) and AlCl₃ (12.0 g) in dichloroethane (60 mL) dropwise over 1 h.After addition was completed, the solution was stirred for an additional1 h at 75° C., then cooled to room temperature. The solution was pouredinto a mixture of crushed ice (100 g) and 2 N HCl (100 mL), at whichpoint a white precipitate formed. The mixture was diluted with ethylacetate (1.8 L), and the organic layer separated. The organic solutionwas washed sequentially with 1 N HCl (500 mL) and saturated brine (500mL), dried over magnesium sulfate, filtered and evaporated to provide anorange powder. The powder was triturated with acetone (80 mL), collectedon a Buchner funnel, rinsed with diethyl ether (80 mL) and dried toprovide a cream colored solid (4.22 g). The product was further purifiedvia recrystallization from ethanol (200 mL) to give colorless plates(3.63 g, 26.0%); mp 263-265° C., with softening at 250° C. (Lit.⁵272-273° C.); ¹ H NMR (DMSO-d₆): 0.95 (3H, t, J=7.4 Hz, CH₃), 1.08 (6H,d, J=6.9 Hz, 2 CH₃), 1.59 (2H, sextet, J=7.4 Hz, CH₂), 2.87 (2H, t,J=7.4 Hz, CH₂), 3.24 (1H, heptet, J=6.9 Hz, CH), 5.93 (1H, s, H₃), 6.37(1H, s, H₆), 11.16 and 11.44 (2H, 2 s, 2 OH); EIMS: 290 (23.2, M⁺), 247(100, M-C₃ H₇), 219 (11.1, M-C₃ H₇ CO); IR (KBr) cm⁻¹ : 3216 (s, OH),1684 (s, C═O); Anal. calcd. for C₁₆ H₁₈ O₅ : C, 66.20; H, 6.25. Found:C, 66.15; H, 6.21.

EXAMPLE 226,6-dimethyl-9-hydroxy-10-isobutyryl-4-propyl-2H,6H-benzo[1,2-b:3,4-b']dipyran-2-one(Scheme V, 13a, R₁ =n-Pr, R₂ =R₇ =H, R₃ =R₄ =R₅ =Me)

To a solution of 12a (2.90 g, 10.0 mmol) in pyridine (5 mL) was added4,4-dimethoxy-2-methylbutan-2-ol (1.49 g, 10.1 mmol), and the solutionheated to ref lux. After heating for 40 h, TLC indicated completeconsumption of starting material. The reaction was cooled to roomtemperature and the pyridine removed in vacuo. The dark colored residuewas dissolved in ethyl acetate (50 mL) and washed sequentially with 2 NHCl (50 mL ×2), 5% NaHCO₃ (50 mL) and saturated brine (50 mL). Thesolution was dried over magnesium sulfate, filtered and evaporated toprovide a dark orange solid, which was chromatographed on a silica gelcolumn (125 g) and eluted with ethyl acetate/hexane (1:4) to afford thepure product as a bright orange crystalline solid (2.51 g, 70.5%); mp70-72° C.; ¹ H NMR (CDCl₃): 1.05 (3H, t, J=7.3 Hz, CH₃), 1.26 (6H, d,J=6.7 Hz, 2 CH₃), 1.54 (6H, s, 2 CH₃), 1.66 (2H, sextet, J=7.7 Hz, CH₂),2.91 (2H, t, J=7.7 Hz, CH₂), 4.06 (1H, heptet, J=6.7 Hz, CH), 5.58 (1H,d, J=9.9 Hz, H₇), 6.01 (1H, s, H₃), 6.73 (1H, d, J=9.9 Hz, H₈), 14.45(1H, s, OH); EIMS: 356 (48.0, M⁺), 341 (100, M-CH₃), 313 (65.0, M-C₃H₇); IR (KBr) cm⁻¹ : 1732; Anal. calcd. for C₂₁ H₂₄ O₅ : C, 70.77; H,6.79. Found: C, 70.73; H, 6.78.

EXAMPLE 23(±)-6,6-Dimethyl-10-(2,2-dimethyl-3-hydroxybutyro)-9-hydroxy-4-propyl-2H,6H-benzo[1,2-b:3,4-b']dipyran-2-one (Scheme V, 14a, R₁ =n-Pr, R₂ =R₇ =R₈=H, R₃ =R₄ =R₅ =R₆ =R₉ =Me)

To a solution of 13a (1.25 g, 3.51 mmol) in anhydrous THF (20 mL) underN₂ at -78° C. was added LDA (2.0 M in heptane/THF/ethyl benzene, 4.39mL, 8.78 mmol) dropwise, and the resulting red solution stirred for 1 h.A solution of acetaldehyde (1.54 g, 35.1 mmol) in THF (6 mL) was addeddropwise, and the reaction mixture stirred at -78° C. for 3 hourswhereupon the reaction was quenched by slowly adding 2.5 M ethanolic HCl(10 mL), and the solution then allowed to warm to room temperature. Thesolvent was evaporated in vacuo and the residue partitioned betweenethyl acetate (100 mL) and saturated NaHCO₃ (100 mL). The organic layerwas collected and washed with saturated brine (100 mL), dried overmagnesium sulfate, filtered and evaporated to provide a brown solid. Theproduct was triturated with ethyl acetate/hexane (1:1, 15 mL), collectedon a Buchner funnel, rinsed with fresh solvent and air dried to give thedesired product as a white powder (654 mg, 46.6%). An analytical samplewas obtained via recrystallization from ethyl acetate/hexane (1:1); mp190-191° C.; ¹ H NMR (CDCl₃): 1.04 (3H, t, J=7.4 Hz, CH₃), 1.25 (3H, s,CH₃), 1.29 (3H, d, J=6.4 Hz, CH₃), 1.33 (3H, s, CH₃), 1.48 (3H, s, CH₃),1.52 (3H, s, CH₃), 1.66 (2H, sextet, J=7.5 Hz, CH₂), 2.39 (1H, broad-s,OH), 2.88 (m, 2H, CH₂), 4.47 (1H, q, J=6.4 Hz, CH), 5.56 (1H, d, J=10.0Hz, H₇), 5.92 (1H, s, H₃), 6.64 (1H, d, J=10.0 Hz, H₈), 8.99 (1H, s,OH): EIMS: 400 (1.1, M⁺), 356 (37.5, M-C₂ H₄ O) , 341 (100, M-CH₃ --C₂H₄ O), 313 (68.2, M-C₃ H₇ --C₂ H₄ O); IR (KBr) cm⁻¹ : 3246 (broad-s,OH), 1686 (s, C═O); Anal. calcd. for C₂₃ H₂₈ O₆ : C, 68.98; H, 7.05.Found. C, 69.03; H, 6.99.

EXAMPLE 24(±)-6.6-Dimethyl-10-(2,3-dimethyl-3-hydroxybutyro)-9-hydroxy-4-propyl-2H,6H-benzo[1,2-b:3,4-b']dipyran-2-one (Scheme V, 14b, R₁ =n-Pr, R₂ =R₃ =R₇=H, R₄ =R₅ =R₆ =R₈ =R₉ =Me)

To a suspension of 4 (1.2 g, 3.50 mmol) in THF (16 mL) at -78° C. wasadded a solution of LDA in heptane/THF/ethyl benzene (2 M, 5.0 mL, 10.0mmol) dropwise under N₂. The solution was stirred at -78° C. for 1 h andacetone (2.0 mL, 27.2 mmol) was added quickly via syringe. The solutionwas stirred at -78° C. for 3 h, quenched with methanolic HCl (2 M, 15mL) at -78° C., then allowed to warm to room temperature. The reactionmixture was concentrated and partitioned between ethyl acetate (150 mL)and saturated NaHCO₃ (100 mL). The organic layer was collected andwashed with saturated brine (50 mL), dried over magnesium sulfate,filtered and concentrated to provide a red oil (1.36 g), an analyticalsample of which was obtained via silica gel column chromatography (ethylacetate/hexane, 1:4) as an off-white solid: mp 99-102° C.; ¹ H NMR(CDCl₃): 1.05 (3H, t, J=7.3 Hz, CH₃), 1.29 (3H, s, CH₃), 1.32 (3H, s,CH₃), 1.39 (3H, d, J=6.8 Hz, CH₃), 1.55 (6H, s, 2 CH₃), 1.67 (2H,sextet, J=7.7 Hz, CH₂), 2.91 (2H, t, J=7.7 Hz, CH₂), 3.52 (1H, broad-s,OH), 4.03 (1H, q, J=6.8 Hz, CH), 5.60 (1H, d, J=9.9 Hz, H₇), 6.03 (1H,s, H₃), 6.73 (1H, d, J=10.1 Hz, H₈), 13.81 (1H, s, OH); EIMS: 401 (5.1,M+1), 400 (21.5, M⁺), 385 (6.2, M-CH₃), 342 (38.9, M-C₃ H₇ O+1), 327(100, M-CH₃ --C₃ H₇ O+1); IR (KBr) cm⁻¹ : 3547 (w, OH), 3449 (vw, broad,OH), 1734 (vs, C═O); Anal. calcd. for C₂₃ H₂₈ O₆ : C, 68.98; H, 7.04.Found: C, 68.98; H, 7.04.

EXAMPLE 25 (±)-syn and(±)-anti-6,6-Dimethyl-9-hydroxy-10-(2-methyl-3-hydroxypentanoyl)-4-propyl-2H,6H-benzo[1,2-b:3,4-b']dipyran-2-one(Scheme V, 14c, R₁ =n=Pr, R₂ =R₃ =R₇ =R₈ =H, R₄ =R₅ =R₆ =Me, R₉ =Et)

To a solution of 4 (1.75 g, 5.11 mmol) in THF (27.0 mL) at -78° C. wasadded dropwise a solution of LDA in heptane/THF/ethyl benzene (2 M, 7.0mL, 14.0 mmol) under N₂. The solution was stirred at -78° C. for 1 h,and propionaldehyde (2.2 mL, 31.2 mmol) was added quickly via syringe.The solution was stirred at -78° C. for 3 h, quenched with methanolicHCl (2 M, 25 mL) at -78° C., then warmed to room temperature. Themixture was extracted with ethyl acetate (350 mL), washed sequentiallywith 150 mL each of saturated NaHCO₃ and saturated brine, dried overmagnesium sulfate, filtered and concentrated to provide a diastereomericmixture of the product as a red oil (2.44 g, 100%), which was notfurther purified and used for the next step.

EXAMPLE 26 (±)-10,11-Dihydro-6,6,10,11,11-pentamethyl-4-propyl-2H,6H,12H-benzo[1,2-b:3,4-b':5,6-b"]tripyran-2,12-dione (Scheme V, 15a, R₁=n-Pr, R₂ =R₇ =R₈ =H, R₃ =R₄ =R₅ =R₆ =R₉ =Me)

To a solution of 14a (0.5 g, 1.25 mmol) and triphenylphosphine (492 mg,1.88 mmol) in THF (10 mL) was added a solution of diethylazodicarboxylate (327 mg, 1.88 mmol) in THF (2 mL) dropwise under N₂.The reaction mixture was stirred for 2.5 h, after which it was pouredinto saturated NH₄ Cl (100 mL). The solution was extracted with ethylacetate (100 mL), and the separated organic layer washed sequentiallywith H₂ O (100 mL) and saturated brine (100 mL). After drying overmagnesium sulfate, the solution was filtered and concentrated in vacuoto provide a yellow oil. Column chromatography through 75 g silica gel(ethyl acetate/hexane, 1:2) provided the desired product as a whitecrystalline solid (449 mg, 94.0%). An analytical sample was obtained viarecrystallization from ethyl acetate/hexane (2:1): mp 157° C.; ¹ H NMR(CDCl₃): 1.03 (3H, t, J=7.3 Hz, CH₃), 1.09 (3H, s, CH₃), 1.19 (3H, s,CH₃), 1.43 (3H, d, J=6.5 Hz, CH₃), 1.53 (3H, s, CH₃), 1.55 (3H, s, CH₃),1.64 (2H, sextet, J=7.7 Hz, CH₂), 2.88 (2H, t, J=7.7 Hz, CH₂), 4.34 (1H,q, J=6.4 Hz, H₁₀), 5.60 (1H, d, J=10.0 Hz, H₇), 6.04 (1H, s, H₃), 6.66(1H, d, J=10.0 Hz, H₈); EIMS: 382 (60.8, M+), 367 (100, M-CH₃), 312(50.3 (M-C₅ H₁₀), 297 (74.5, M-CH₃ -C₅ H₁₀); IR (KBr) cm⁻¹ : 1730 (vs,C═O); Anal. calcd. for C₂₃ H₂₆ O₅ : C, 72.23; H, 6.85. Found: C, 72.35;H, 6.90.

EXAMPLE 27 (±)-10,11-Dihydro-6,6,10,10,11-pentamethyl-4-propyl-2H, 6H,12H-benzo[1,2-b:3,4-b':5,6-b"]tripyran-2,12-dione (Scheme V, 15b, R₁=n-Pr, R₂ =R₃ =R₇ =H, R₄ =R₅ =R₆ =R₈ =R₉ =Me)

To a solution of crude 14b (980 mg, 2.19 mmol) and triphenylphosphine(859.0 mg, 3.28 mmol) in THF (15 mL) was slowly added diethylazodicarboxylate (DEAD, 0.50 mL, 3.17 mmol) under N₂. The red solutionwas stirred for 2.5 h at room temperature, then quenched with saturatedNH₄ Cl (10 mL). The solution was extracted with ethyl acetate (200 mL),washed sequentially with 50 mL each of H₂ O and saturated brine, driedover magnesium sulfate, filtered and concentrated to provide a yellowresidue (2.37 g). Purification by silica gel column chromatography(ethyl acetate/hexane, 1:10) provided, after overnight drying under highvacuum in the presence of P₂ O₅, the desired product as an off-whitesolid (373.7 mg, 44.6%): mp 140-141° C.; ¹ H NMR (CDCl₃): 1.03 (3H, t,J=7.3 Hz, CH₃), 1.19 (3H, d, J=7.0 Hz, CH₃), 1.34 (3H, s, CH₃), 1.53(6H, s, 2 CH₃), 1.55 (3H, s, CH₃), 1.65 (2H, sextet, J=7.8 Hz, CH₂),2.72 (1H, q, J=7.0 Hz, H₁₁), 2.85-2.91 (2H, m, CH₂), 5.60 (1H, d, J=10.1Hz, H₇), 6.03 (1H, s, H₃), 6.65 (1H, d, J=10.0 Hz, H₈); EIMS: 382 (61.2,M⁺), 367 (82.0, M-CH₃), 312 (46.0, M-C₅ H₁₀), 297 (100, M-CH₃ --C₅ H₁₀);IR (KBr) cm⁻¹ : 1728 (vs, C═O); Anal. calcd. for C₂₃ H₂₆ O₅ : C, 72.23;H, 6.85. Found: C, 71.95; H, 6.88.

EXAMPLE 28(±)-10,11-trans-10,11-Dihydro-10-ethyl-4-propyl-6,6,11-trimethyl-2H, 6H,12H-benzo[1,2-b:3,4-b"]tripyran-2,12-dione (15c) and(±)-10,11-cis-10,11-dihydro-10-ethyl-4-propyl-6,6,11-trimethyl-2H,6H,12H-benzo[1,2-b:3,4-b':5,6-b"]tripyran-2,12-dione(15d, Scheme V)

To a solution of 14c (2.44 g, 5.11 mmol) and triphenylphosphine (1.96mg, 7.48 mmol) in THF (30.0 mL) was slowly added diethylazodicarboxylate (DEAD, 1.16 mL, 7.37 mmol) under N₂. The red solutionwas stirred for 2.5 h at room temperature, then quenched with saturatedNH₄ Cl (22 mL). The solution was warmed to room temperature andextracted with ethyl acetate (400 mL), washed with H₂ O (100 mL) andbrine (100 mL) and dried over magnesium sulfate. After filtration, thesolution was concentrated in vacuo to provide a yellow residue (5.75 g).The crude product was purified by repetitive silica gel columnchromatography (3X) using ethyl acetate/hexane (1:4.5) as eluent. Thedesired fractions were combined, concentrated in vacuo and dried underhigh vacuum overnight in the presence of P₂ O₅ to afford 15c (765.4 mg,39.2%) and 15d (350.4 mg, 17.9%).

15c (R₁ =n-Pr, R₂ =R₄ =R₇ =R₈ =H, R₃ =R₅ =R₆ =Me, R₉ =Et): mp 155-158°C.; ¹ H NMR (CDCl₃): 1.03 (3H, t, J=7.4 Hz, CH₃), 1.13 (3H, t, J=7.4 Hz,CH₃), 1.22 (3H, d, J=6.9 Hz, CH₃), 1.53 (3H, s, CH₃), 1.56 (3H, s, CH₃),1.64 (2H, sextet, J=7.6 Hz, CH₂), 1.78-1.95 (2H, m, CH₂), 2.62 (1H, dq,J=10.4 Hz, J=7.0 Hz, H₁₁), 2.88 (2H, t, J=7.7 Hz,CH₂), 4.14 (1H, ddd,J=3.5 Hz, J=7.8 Hz, J=10.7 Hz, H₁₀), 5.61 (1H, d, J=10.0 Hz, H₇), 6.04(1H, s, H₃), 6.66 (1H, d, J=10.0 Hz, H₈); EIMS: 382 (37.2, M⁺), 367(100, M-CH₃), 297 (47.2, M-CH₃ --C₅ H₁₀); IR (KBr) cm⁻¹ : 1738 (vs, C═O); Anal. calcd. for C₂₃ H₂₆ O₅ : C, 72.23; H, 6.85. Found: C, 71.75; H,7.02.

15d (R₁ =n-Pr, R₂ =R₃ =R₇ =R₈ H, R₄ =R₅ =R₆ =Me, R₉ =Et): mp 100-102°C.; ¹ H NMR (CDCl₃): 1.03 (3H, t, J=7.3 Hz, CH₃), 1.07 (3H, t, J=7.4 Hz,CH₃), 1.14 (3H, d, J=7.3 Hz, CH₃), 1.54 (3H, s, CH₃), 1.55 (3H, CH₃),1.65 (2H, sextet, J=7.6 Hz, CH₂), 1.83-1.98 (2H, m, CH₂), 2.70 (1H, dq,J=3.2 Hz, J=7.3 Hz, H₁₁), 2.88 (2H, t, J=7.6 Hz, CH₂), 4.39 (1H, ddd,J=3.4 Hz, J=5.3 Hz, J=8.8 Hz, H₁₀), 5.60 (1H, d, J=10.0 Hz, H₇), 6.05(1H, s, H₃), 6.66 (1H, d, J=10.0 Hz, H₈); EIMS: 382 (55.0, M⁺), 367(100, M-CH₃), 297 (52.7, M-CH₃ --C₅ H₁₀); IR (KBr) cm⁻¹ : 1732 (vs,C═O); Anal. calcd. for C₂₃ H₂₆ O₅ : C, 72.23; H, 6.85. Found: C, 71.80;H, 6.97.

EXAMPLE 29(±)-10,12-cis-10,11-Dihydra-12-hydroxy-6,6,10,11,11-pentamethyl-4-propyl-2H,6H, 12H-benzo [1,2-b:3,4-b ':5,6-b"] tripyran-2-one (16a) and(±)-10,12-trans-10,11-dihydro-12-hydroxy-6,6,10,11,11-pentamethyl-4-propyl-2H,6H, 12H-benzo [1,2-b:3,4-b':5,6-b"]tripyran-2-one (16b, Scheme V)

To a solution of 15a (252 mg, 0.661 mmol) in ethanol/THF (1:1, 8 mL) wasadded sodium borohydride (25.1 mg, 0.661 mmol) and the solution stirredat room temperature for 30 minutes. The reaction was quenched by theaddition of water (1 mL), and the solvent then removed in vacuo. Theresidue was partitioned between 20 mL each of ethyl acetate and 1 M HCl,and the organic phase separated and washed sequentially with 5% NaHCO₃and saturated brine. After drying over magnesium sulfate, the solutionwas evaporated to give the product as a pale-yellow foam. TLC analysis(ethyl acetate/hexane, 1:2) showed the two epimeric alcohols 16a and 16bat R_(f) 0.30 and 0.25, as well as a minor impurity at Rf 0.55.Separation via column chromatography (75 g silica gel, ethylacetate/hexane, 1:2) provided 16a (127.7 mg, 50.3%) and 16b (18.8 mg,7.4%) as a white foam and an off-white waxy solid, respectively.

16a (R₁ =n-Pr, R₂ =R₇ =R₈ =H, R₃ =R₄ =R₅ =R₆ =R₉ =Me): ¹ H NMR (CDCl₃):1.04 (3H, t, J=7.3 Hz, CH₃), 1.06 (6H, s, 2 CH₃), 1.40 (3H, d, J=6.7 Hz,CH₃), 1.47 (3H, s, CH₃), 1.50 (3H, s, CH₃), 1.66 (2H, sextet, J=7.3 Hz,CH₂), 2.80-2.99 (2H, m, CH₂), 3.39 (1H, d, J=3.2 Hz, OH), 3.99 (1H, q,J=6.7 Hz, H₁₀), 4.70 (1H, d, J=3.2 Hz, H₁₂), 5.54 (1H, d, J=9.9 Hz, H₇),5.94 (1H, s, H₃), 6.63 (1H, d, J=9.9 Hz, H₈); EIMS: 384 (59.0, M⁺), 369(100, M-CH₃), 314 (44.7, M-C₅ H₁₀), 299 (88.8, M-CH₃ --C₅ H₁₀); IR (KBr)cm⁻¹ : 3432 (broad-s, OH), 1734 (vs, C═O); Anal. calcd. for C₂₃ H₂₈ O₅ :C, 71.85; H, 7.34. Found: C, 71.74; H, 7.43.

16b (R₁ =n-Pr, R₂ =R₇ =R₈ =H, R₃ =R₄ =R₅ =R₆ =R₉ =Me): ¹ H NMR (CDCl₃):0.78 (3H, s, CH₃), 1.04 (3H, t, J=7.3 Hz, CH₃), 1.11 (3H, s, CH₃), 1.36(3H, d, J=6.5 Hz, CH₃), 1.49 (6H, s, 2 CH₃), 1.64 (2H, m, CH₂), 2.47(1H, broad-s, OH), 2.89 (2H, m, CH₂), 4.35 (1H, q, J=6.5 Hz, H₁₀), 4.63(1H, broad-s, H₁₂), 5.54 (1H, d, J=9.8 Hz, H₇), 5.96 (1H, s, H₃), 6.65(1H, d, J=9.8 Hz, H₈); EIMS: 384 (40.7, M⁺), 369 (100, M-CH₃), 314(13.5, M-C₅ H₁₀), 299 (48.4, M-CH₃ --C₅ H₁₀); Anal. calcd. for C₂₃ H₂₈O₅ : C, 71.85; H, 7.34. Found: C, 71.79; H, 7.49.

EXAMPLE 30(±)-11,12-cis-10,11-Dihydro-12-hydroxy-6,6,10,10,11-pentamethyl-4-propyl-2H,6H, 12H-benzo[1,2-b:3,4-b':5,6-b"]tripyran-2-one (16c) and(±)-11,12-trans-10,11-dihydro-12-hydroxy-6,6,10,10,11-pentamethyl-4-propyl-2H,6H, 12H-benzo [1,2-b:3,4-b':5,6-b"]tripyran-2-one (16d, Scheme V)

To a solution of 15b (289.7 mg, 0.75 mmol), triphenylphosphine oxide(927.0 mg, 3.33 mmol) and CeCl₃ (H₂ O)₇ (842.0 mg, 2.25 mmol) in ethanol(15 mL) at 0° C. was slowly added NaBH₄ (195.0 mg, 5.15 mmol) under N₂.The suspension was stirred for 1 h at room temperature, then quenchedwith saturated NH₄ Cl (30 mL). The solution was extracted with ethylacetate (200 mL), washed with brine (50 mL), dried over magnesiumsulfate, filtered and concentrated to afford a pink crystalline solid(1.38 g). Silica gel column chromatography (ethyl acetate/hexane, 1:5)provided 16c (100.0 mg, 34.3%) as off-white foam and 16d which wasfurther purified by preparative TLC (silica gel, diethyl ether/hexane,2:1) as off-white foam (56.0 mg, 19.2%).

16c (R₁ =n-Pr, R₂ =R₃ =R₇ =H, R₄ =R₄ =R₆ =R₈ =R₉ =Me): mp 44-45° C.; ¹ HNMR (CDCl₃): 1.04 (3H, t, J=7.3 Hz, CH₃), 1.24 (3H, d, J=7.1 Hz, CH₃),1.38 (3H, s, CH₃), 1.45 (3H, s, CH₃), 1.47 (3H, s, CH₃), 1.51 (3H, s,CH₃), 1.66 (2H, sextet, J=7.3 Hz, CH₂), 1.96-2.04 (1H, m, H₁₁), 2.8-3.0(2H, m, CH₂), 3.02 (1H, d, J=4.0 Hz, OH), 4.94 (1H, t, J=4.2 Hz, H₁₂),5.53 (1H, d, J=10.0 Hz, H₇), 5.94 (1H, s, H₃), 6.65 (1H, d, J=9.9 Hz,H₈); EIMS: 385 (22.1, M+1), 384 (61.8, M⁺), 369 (71.1, M-CH₃), 351(29.5, M-CH₃ --H₂ O), 299 (100, M-CH₃ --C₅ H₁₀); IR (KBr) cm⁻¹ : 3451(broad-m, OH), 1709 (s, C═O); Anal. calcd. for C₂₃ H₂₈ O₅ : C, 71.85; H,7.33. Found: C, 71.63; H, 7.64.

16d (R₁ =n-Pr, R₂ =R₄ =R₇ =H, R₃ =R₅ =R₆ =R₈ =R₉ =Me): mp 40-42° C.; ¹ HNMR (CDCl₃): 1.04 (3H, t, J=7.3 Hz, CH₃), 1.13 (3H, d, J=7.0 Hz, CH₃),1.21 (3H, s, CH₃), 1.46 (3H, s, CH₃), 1.48 (3H, s, CH₃), 1.52 (3H, s,CH₃), 1.67 (2H, sextet, J=7.6 Hz, CH₂), 2.03 (1H, quintet, J=7.2 Hz,H₁₁), 2.8-3.0 (2H, m, CH₂), 3.66 (1H, s, OH), 4.69 (1H, d, J=7.4 Hz,H₁₂), 5.54 (1H, d, J=10.0 Hz, H₇), 5.94 (1H, s, H₃), 6.63 (1H, d, J=9.9Hz, H8); EIMS: 385 (8.7, M+1), 384 (36.0, M⁺), 369 (65.8, M-CH₃), 351(17.6, M-CH₃ -H₂ O), 299 (100, M-CH₃ --C₅ H₁₀); IR (KBr) cm⁻¹ : 3437 (w,OH), 1734 (s, C═O); Anal. calcd. for C₂₃ H₂₈ O₅ : C, 71.85; H, 7.33.Found: C, 71.70; H, 7.56.

EXAMPLE 31(±)-10,11-trans,11,12-cis-10,11-Dihydro-10-ethyl-12-hydroxy-4-propyl-6,6,11-trimethyl-2H,6H,12H-benzo[1,2-b:3,4-b':5,6-b"]tripyran-2-one(16e) and(±)-10,11-trans-11,12-trans-10,11-dihydro-10-ethyl-12-hydroxy-4-propyl-6,6,11-trimethyl-2H,6H,12H-benzo[1,2-b:3,4-b':5,6-b"]tripyran-2-one(16f, Scheme V)

To a suspension of 15c (454.7 mg, 1.19 mmol), triphenylphosphine oxide(1.38 g, 4.96 mmol) and CeCl₃ (H₂ O)₇ (1.21 g, 3.25 mmol) in ethanol (10mL) at 0° C. was slowly added NaBH₄ (312.0 mg, 8.25 mmol) under N₂. Thesuspenion was stirred for 3 h at room temperature. The reaction mixturewas quenched with saturated NH₄ Cl (15 mL), extracted with ethyl acetate(100 mL ×3), washed with brine (50 mL), dried over magnesium sulfate,filtered and concentrated to provide pink crystals (1.97 g). Silica gelcolumn chromatography (ethyl acetate/hexane, 1:4) afforded a yellow oil,which consisted of mixture of 16e and 16f (261.0 mg). The compounds wereseparated using preparative HPLC (normal phase, ethyl acetate/hexane,3:7). The desired fractions were combined and concentrated in vacuo anddried overnight under high vacuum in the presence of P₂ O₅ to afford 16e(yellow oil, 46.5 mg, 10.1%) and 16f (white solid, 137.6 mg, 30.1%).

16e (R₁ =n-Pr, R₂ =R₄ =R₇ =R₈ =H, R₃ =R₅ =R₆ =Me, R₉ =Et): ¹ H NMR(CDCl₃): 1.03 (3H, t, J=7.3 Hz, CH₃), 1.10 (3H, t, J=7.6 Hz, CH₃). 1.13(3H, d, J=6.8 Hz, CH₃), 1.48 (3H, s, CH₃), 1.49 (3H, s, CH₃), 1.65 (2H,sextet, J=7.4 Hz, CH₂), 1.76-1.98 (3H, m, CH₂ +H₁₁), 2.80-2.92 (3H, m,CH₂ +OH), 4.10 (1H, ddd, J=2.9 Hz, J=7.9 Hz, J=10.7 Hz, H₁₀), 4.98 (1H,d,J=3.3 Hz, H₁₂), 5.54 (1H, d, J=9.9 Hz, H₇), 5.94 (1H, s, H₃), 6.63(1H, d, J=9.9 Hz, H₈); EIMS: 385(10.5, M+1), 384 (35.8,M⁺), 369 (78.4,M-CH₃), 366 (43.1, M-H₂ O), 351 (39.0, M-CH₃ -H₂ O), 337 (100, M-H₂O--C₂ H₅), 299 (37.7, M-CH₃ --C₅ H₁₀); IR (neat, thin film) cm⁻¹ : 3432(w, OH), 1709 (s, C═O); Anal. calcd. for C₂₃ H₂₈ O₅.1/4 H₂ O: C, 71.02;H, 7.38. Found: C, 71.10; H, 7.40.

16f (R₁ =n-Pr, R₂ =R₄ =R₇ =R₈ =H, R₃ =R₅ =R₆ =Me, R₉ =Et): mp 103-105°C.; ¹ H NMR (CDCl₃): 1.04 (3H, t, J=7.3 Hz, CH₃), 1.07 (3H, t, J=7.4 Hz,CH₃), 1.13 (3H, d, J=6.9 Hz, CH₃), 1.47 (3H, s, CH₃), 1.51 (3H, s, CH₃),1.66 (2H, sextet, J=7.6 Hz, CH₂), 1.79-1.90 (2H, m, CH₂), 2.05 (1H, m,H₁₁), 2.90 (2H, m, CH₂), 3.53 (1H, s, OH), 3.78 (1H, dt, J=4.1 Hz, J=8.1Hz, H₁₀), 4.73 (1H, d, J=6.7 Hz, H₁₂), 5.54 (1H, d, J=10.0 Hz, H₇), 5.95(1H, s, H₃), 6.63 (1H, d, J=9.9 Hz, H₈); EIMS: 385 (7.6, M+1), 384(31.1, M⁺), 369 (100, M-CH₃), 351 (9.5, M-CH₃ --H₂ O), 337 (11.5, M-H₂O--C₂ H₅), 299 (36.9, M-CH₃ --C₅ H₁₀); IR (KBr) cm⁻¹ : 3493, 3435 and3250 (w, OH), 1699 (s, C═O); Anal. calcd. for C₂₃ H₂₈ O₅ : C, 71.85; H,7.33. Found: C, 71.46; H, 7.34.

EXAMPLE 32(±)-10,11-cis-11,12-trans-10,11-Dihydro-10-ethyl-12-hydroxy-4-propyl-6,6,11-trimethyl-2H,6H,12H-benzo[1,2-b:3,4-b':5,6-b"]tripyran-2-one(16 g) and(±)-10,11,12-cis-10,11-dihydro-10-ethyl-12-hydroxy-4-propyl-6,6,11-trimethyl-2H,6H,12H-benzo[1,2-b:3,4-b':5,6-b"]tripyran-2-one(16h, Scheme V)

To a solution of 15d (290.5 mg,0.76 mmol) in ethanol (15 mL) at 25° C.was added NaBH₄ (269.0 mg, 7.11 mmol) portionwise under N₂. Thesuspension was stirred for 1 h at room temperature, then quenched withsaturated NH₄ Cl (6 mL). The solution was extracted with ethyl acetate(200 mL), washed with brine (80 mL), dried over magnesium sulfate,filtered and concentrated to provide a pink residue (455.8 mg). Thecrude product was purified by silica gel preparative TLC (ethylacetate/hexane, 2:1). The desired bands were scraped, combined,extracted, concentrated in vacuo and dried under high vacuum overnightin the presence of P₂ O₅ to afford the desired products 16g (229 mg, 78%yield) with 95% purity as indicated by HPLC) and 16h (55.9 mg, 19%yield) with 92% purity. The analytical samples were obtained by furtherpurification via preparative HPLC (normal phase, ethyl acetate/hexane,3:7).

16g (R₁ =n-Pr, R₂ =R₃ =R₇ =R₈ =H, R₄ =R₅ =R₆ =Me, R₉ =Et): ¹ H NMR(CDCl₃): 1.03 (3H, t, J=7.4 Hz, CH₃), 1.04 (3H, t, J=7.3 Hz, CH₃),1.12(3H, d, J=7.1 Hz, CH₃), 1.49 (3H, s, CH₃), 1.66 (2H, sextet, J=7.3 Hz,CH₂), 1.8-2.0 (2H, m, CH₂), 2.3-2.4 (1H, m, H₁₁), 2.8-3.0 (2H, m, CH₂),3.30 (1H, s, OH), 4.06 (1H, dt, J=10.1 Hz, J=3.3 Hz, H₈), 5.10 (1H, d,J=5.2 Hz, H₁₂), 5.55 (1H, d, J=10.0 Hz, H₇), 5.94 (1H, s, H₃), 6.63 (1H,d, J=10.0 Hz, H₈); EIMS: 385 (6.3, M+1), 384 (27.3, M⁺), 369 (100,M-CH₃), 351 (6.8, M-CH₃ --H₂ O), 337 (4.2, M-H₂ O--C₂ H₅), 299 (34.7,M-CH₃ --C₅ H₁₀); IR (KBr) cm⁻¹ : 3449 (m, OH), 1734 (vs, C═O); Anal.calcd. for C₂₃ H₂₈ O₅ : C, 71.85; H, 7.33. Found: C, 71.79; H, 7.39.

16h (R₁ =n-Pr, R₂ =R₃ =R₇ =R₈ =H, R₄ =R₅ =R₆ =Me, R₉ =Et): ¹ H NMR(CDCl₃): 0.79 (3H, d, J=7.3 Hz, CH₃), 1.04 (3H, t, J=7.3 Hz, CH₃), 1.11(3H, t, J=7.3 Hz, CH₃), 1.49 (3H, s, CH₃), 1.51 (3H, s, CH₃), 1.67 (2H,sextet, J=7.4 Hz, CH₂), 1.92 (2H, m, CH₂), 2.10 (1H, tq, J=2.0 Hz, J=7.3Hz, H₁₁), 2.79 (1H, s, OH), 2.81-2.90 (2H, m, CH₂), 4.23 (1H, ddd, J=1.9Hz, J=5.4 Hz, J=8.7 Hz, H₁₀), 4.87 (1H, d, J=19 Hz, H₁₂), 5.54 (1H, d,J=10.0 Hz, H₇), 5.96 (1H, s, H₃), 6.66 (1H, d, J=9.9 Hz, H₈); EIMS: 385(6.1, M+1), 384 (26.0, M⁺), 369 (100, M-CH₃), 351 (9.8, M-CH₃ --H₂ O),337 (8.2, M-H₂ O--C₂ H₅), 299 (17.6, M-CH₃ --C₅ H₁₀); IR (neat, thinfilm) cm⁻¹ : 3410 (w, OH), 1732 (s, C═O).

EXAMPLE 33(±)-10,11-trans-4-Propyl-7,8,10,11-tetrahydro-6,6,10,11-tetramethyl-2H,6H, 12H-benzo[1,2-b:3,4-b':5,6-b"]tripyran-2,12-dione (Scheme VI, 18a,R₁ =n-Pr, R₂ =R₄ =R₇ =R₈ =H, R₃ =R₅ =R₆ =R₉ =Me)

To a solution of (±)-7 (534 mg, 1.45 mmol) in ethanol/methylene chloride(1:1, 50 mL, Parr apparatus) under N₂ was added 10% palladium/carbon(53.4 mg) at ambient temperature. The mixture was shaken under hydrogen(2 atm) for 1 h, then gravity filtered through Whatmann filter paper.The solvent was evaporated to give a white crystalline solid which wasfiltered through a short plug of silica gel, eluting with methylenechloride/methanol (97:3). The pure compound (±)-18a (441 mg, 82.2%) wasobtained as white plates by recrystallization from ethyl acetate: mp165° C.; ¹ H NMR (CDCl₃): 1.01 (3H, t, J=7.3 Hz, CH₃), 1.21 (3H, d,J=6.8 Hz, CH₃), 1.42 (3H, s, CH₃), 1.44 (3H, s, CH₃), 1.53 (3H, d, J=6.2Hz, CH₃), 1.61 (2H, sextet, J=7.5 Hz, CH₂), 1.84 (2H, apparent dt, J=2.4Hz, J=6.7 Hz, CH₂), 2.53 (1H, dq, J=11.2 Hz, J=6.8 Hz, H₁₁), 2.69 (2H,apparent dt, J=3.4 Hz, J=6.7 Hz, CH₂), 2.88 (2H, t, J=7.5 Hz, CH₂), 4.28(1H, dq, J=11.2 Hz, J=6.2 Hz, H₁₀), 6.02 (1H, s, H₃); EIMS: 371 (40.8,M+1); 370 (100, M⁺), 314 (99.3, M-C₄ H₈), 299 (21.6, M-C₅ H₁₀ -1), 286(65.0, M-CH₃ --C₅ H₁₀ +1), 271 (20.5, M-CH₃ --C₅ H₈ O), 259 (47.5, M-C₄H₈ --C₃ H₄ O+1); IR (KBr) cm⁻¹ : 1740 (vs, C═O); Anal. calcd. for C₂₂H₂₆ O₅ : C, 71.33; H, 7.07. Found: C, 71.00; H, 7.22.

EXAMPLE 34(±)-10,11-trans-10,11-Dihydro-4-propyl-6,6,10,11-tetramethyl-2H, 6H,12H-benzo[1,2-b:3,4-b':5,6-b"]tripyran-2,12-dione-12-oxime (Scheme VI,19a, R₁ =n-Pr, R₂ =R₄ =R₇ =R₈ =H, R₃ =R₅ =R₆ =R₉ =Me, R₁₀ =H)

Into a 100 mL one-necked round-bottom flask was placed (±)-7 (1.47 g,4.00 mmol) and NH₂ OHHCl (1.39 g, 20.0 mmol). To this mixture was addedmethanol (60 mL), and the solution heated to reflux with magneticstirring until the ketone dissolved. Solid K₂ CO₃ powder (1.38 g, 10.0mmol) was then carefully added, and the reaction allowed to stir atreflux for 4 hours. The solution was cooled at room temperature,filtered to remove the K₂ CO₃ and evaporated in vacuo, to provide ayellow solid. The residue was partitioned between 150 mL each of H₂ Oand ethyl acetate. The organic phase was collected and washedsequentially with 1 N HCl and saturated brine, then dried over magnesiumsulfate, filtered and evaporated to afford a thick yellow syrup, whichwas purified via silica gel column chromatography (75 g), eluting withmethylene chloride/methanol (97:3) to afford the desired product as awhite solid (657 mg, 43%). An analytical sample was obtained viarecrystallization from acetone/hexane (1:3) as colorless prisms; mp200-201° C.; ¹ H NMR (CDCl₃): 1.04 (3H, t, J=7.4 Hz, CH₃), 1.23 (3H, d,J=7.0 Hz, CH₃), 1.33 (3H, d, J=6.5 Hz, CH₃), 1.51 (3H, s, CH₃), 1.54(3H, s, CH₃), 1.67 (2H, sextet, J=7.4 Hz, CH₂), 2.82-3.01 (2H, m, CH₂),3.79 (1H, dq, J=2.0 Hz, J=7.0 Hz, H₁₁), 4.46 (1H, dq, J=2.0 Hz, J=6.5Hz, H₁₀), 5.57 (1H, d, J=9.9 Hz, H₇), 6.02 (1H, s, H₃), 6.67 (1H, d,J=9.9 Hz, H₈), 9.46 (1H, broad-s, OH); EIMS: 384 (12.9,M+1), 383 (49.22,M⁺), 368 (100, M-CH₃), 366 (21.1, M-OH), 352 (15.2, M-NOH); IR (KBr)cm⁻¹ : 3223 (broad, OH), 1740 (C═O); Anal. calcd. for C₂₂ H₂₅ NO₅. 1/4H₂ O): C, 68.11; H, 6.63; N, 3.61. Found: C, 68.40; H, 6.59; N, 3.58.

EXAMPLE 35(±)-10,11-trans-10,11-Dihydro-4-propyl-6,6,10,11-tetramethyl-2H, 6H,12H-benzo[1,2-b:3,4-b':5,6-b"]tripyran-2,12-dione-12-methoxime (SchemeVI, 19b, R₁ =n-Pr, R₂ =R₄ =R₇ =R₈ =H, R₃ =R₅ =R₆ =R₉ =Me, R₁₀ =Me)

Into a one-necked 100 mL round-bottom flask was placed (±)-7 (1.47 g,4.00 mmol) and NH₂ OCH₃ HCl (1.67 g, 20.0 mmol). To this mixture wasadded methanol (60 mL), and the solution heated to reflux with magneticstirring until the ketone dissolved. Solid K₂ CO₃ powder (1.38 g, 10.0mmol) was then carefully added, and the reaction allowed to stir atreflux for 4 hours. The solution was cooled to room temperature,filtered to remove the K₂ CO₃ and evaporated in vacuo, to provide ayellow oil. The residue was partitioned between 150 mL H₂ O and 150 mLethyl acetate. The organic phase was collected and washed sequentiallywith 1 N HCl and saturated brine, then dried over magnesium sulfate,filtered and evaporated to afford a thick yellow syrup. The product waspurified via silica gel column chromatography (75 g), eluting with ethylacetate/hexane (1:3) to afford the desired product as a faintly yellowoil which, upon standing, formed a white solid (598 mg, 38%). Ananalytical sample was obtained via recrystallization from acetone/hexane(1:3) as white plates; mp 143-144° C.; ¹ H NMR (CDCl₃): 1.01 (3H, t,J=7.3 Hz, CH₃), 1.16 (3H, d, J=7.0 Hz, CH₃), 1.28 (3H, d, J=6.4 Hz,CH₃), 1.49 (3H, s, CH₃), 1.50 (3H, s, CH₃), 1.64 (2H, sextet, J=7.3 Hz,CH₂), 2.79-2.99 (2H, m, CH₂), 3.57 (H, dq, J=1.9 Hz, J=7.0 Hz, H₁₁),4.06 (3H, s, OCH₃), 4.37 (1H, dq, J=1.9 Hz, J=6.4 Hz, H₁₀), 5.54 (1H, d,J=10.0 Hz, H₇), 6.00 (1H, s, H₃), 6.62 (1H, d, J=10.0 Hz, H₈); EIMS: 397(61.2, M⁺), 382 (100, M-CH₃), 366 (12.9, M-OCH₃); IR (KBr) cm⁻¹ : 1728(vs, C═O); Anal. calcd. for C₂₃ H₂₇ NO₅ : C, 69.50; H, 6.85; N, 3.52.Found: C, 69.39; H, 6.90; N, 3.59.

EXAMPLE 36 Conversion of (-)-Calanolide A into (-)-Calanolide B

To a solution of (-)-calanolide A (341 mg, 0.922 mmol) in anhydrousmethylene chloride (5 mL) at -78° C. under N₂ was added a solution ofdiethylamidosulfur trifluoride (DAST, 178 mg, 1.11 mmol) in methylenechloride (1 mL) and the resulting yellow solution stirred at -78° C. for4 hours. The reaction was quenched with 0.5 mL methanol, then allowed towarm to room temperature. The solution was diluted with methylenechloride (20 mL), then washed with water (50 mL) and saturated brine (50mL). After drying over magnesium sulfate, the solution was filtered andevaporated to provide a light yellow solid. TLC analysis (silica gel, 3%methanol in methylene chloride) showed two components, one fast-movingand one slow. The material was chromatographed through 80 g silica gel,eluting with 1% methanol in CH₂ Cl₂, and the fractions containing therespective components combined and evaporated to afford 198 mg (61%yield) of compound 22 and 75.3 mg (22%) of (-)-calanolide B.

10(S)-4-propyl-6,6,10,11-tetramethyl-2H,6H,10H-benzo[1,2-b:3,4-b':5,6-b"]tripyran-2-one(22)

¹ H NMR (CDCl₃): 1.03 (3H, t, J=7.4 Hz, CH₃), 1.39 (3H, d, J=6.6 Hz,CH₃), 1.47 (3H, s, CH₃). 1.51 (3H, s, CH₃), 1.66 (2H, sextet, J=7.4 Hz,CH₂), 1.85 (3H, s, CH₃), 2.88 (2H, m, CH₂), 4.89 (1H, q, J=6.6 Hz, H₁₀),5.55 (1H, d, J=10.0 Hz, H₇), 5.93 (1H, s, H₃), 6.62 (1H, d, J=10.0 Hz,H₈), 6.64 (1H, s, H₁₂); EIMS: 353 (15.5, M+1), 352 (53.2, M⁺), 337 (100,M-CH₃). IR (KBr) cm⁻¹ : 1724 (s, C═O); Anal. calcd. for C₂₂ H₂₄ O₄ : C,74.98; H, 6.86. Found: C, 74.87; H, 7.00.

10(S),11(R),12(5)-10,11-Dihydro-12-hydroxy-4-propyl-6,6,10,11-tetramethyl-2H,6H-benzo[1,2-b:3,4-b':5,6-b"]tripyran-2-one[(-)-Calanolide B]

¹ H NMR (CDCl₃): 1.03 (3H, t, J=7.3 Hz, CH₃), 1.14 (3H, d, J=7.0 Hz,CH₃), 1.43 (3H, d, J=6.4 Hz, CH₃), 1.48 (3H, s, CH₃), 1.49 (3H, CH₃),1.66 (2H, sextet, J=7.6 Hz, CH₂), 1.72-1.79 (1H, m, H₁₁), 2.60 (1H, d,J=3.8 Hz, OH), 2.89 (2H, m, CH₂), 4.26 (1H, dq, J=10.7 Hz, 6.3 Hz, H₁₀),4.97 (1H, J=3.8 Hz, H₁₂), 5.53 (1H, d, J=10.0 Hz, H₇), 5.95 (1H, s, H₃),6.63 (1H, d, J=10.0 Hz, H₈); EIMS: 370 (31.1, M⁺), 355 (100, M-CH₃), 299(29.7, M-CH₃ --C₄ H₈); IR (KBr) cm⁻¹ : 3478 (s, sharp, OH), 1703 (S,C═O).

EXAMPLE 37 In Vitro evaluation of (+)-, (±)- and (-)-calanolide A

This example illustrates the anti-HIV viral activity of the synthetic(±) -calanolide A and its pure enantiomers, (+)-calanolide A and(-)-calanolide A, which were evaluated using the publishedMTT-tetrazolium method¹⁸. Retroviral agents AZT and DDC were used ascontrols for comparison purposes.

The cells used for screening were the MT-2 and the humanT4-lymphoblastoid cell line, CEM-SS, and were grown in RPMI 1640 mediumsupplemented with 10% fetal (v/v) heat-inactivated fetal calf serum andalso containing 100 units/mL penicillin, 100 Mg/mL streptomycin, 25 mMHEPES and 20 μg/mL gentamicin. The medium used for dilution of drugs andmaintenance of cultures during the assay was the same as above. TheHTLV-IIIB and HTLV-RF were propagated in CEM-SS. The appropriate amountsof the pure compounds for anti-HIV evaluations were dissolved in DMSO,then diluted in medium to the desired initial concentration. Theconcentrations (μg drug/mL medium) employed were 0.0032 μg/mL; 0.001μg/mL; 0.0032 μg/mL; 0.01 μg/mL; 0.032 μg/mL; 0.1 μg/mL; 0.32 μg/mL; 1μg/mL; 3.2 μg/mL; 10 μg/mL; 32 μg/mL; and 100 μg/mL. Each dilution wasadded to plates in the amount of 100 μL/well. Drugs were tested intriplicate wells per dilution with infected cells while in duplicatewells per dilution with uninfected cells for evaluation of cytotoxicity.On day 6 (CEM-SS cells) and day 7 (MT-2 cells) post-infection, theviable cells were measured with a tetrazolium salt, MTT (5 mg/mL), addedto the test plates. A solution of 20% SDS in 0.001 N HCl is used todissolve the MTT formazan produced. The optical density value was afunction of the amount of formazan produced which was proportional tothe number of viable cells. The percent inhibition of CPE per drugconcentration was measured as a test over control and expressed inpercent (T/C%). The data is summarized in FIGS. 1(a-e), 2(a-e), 3(a-e),4(a-d), and 5(a-d).

FIGS. 1(a) to 1(e) illustrate in vitro MTT assay results using anisolate, G910-6 HIV viral strain¹⁹, which is AZT-resistant. The datashows that (-)-calanolide A was relatively non-toxic at concentrationsof 1 μg/mL but exhibited very little antiviral effect. Moreover,(±)-calanolide A and (+)-calanolide A were effective in reducing viralCPE. As expected, AZT had little to no effect in reducing viral CPE andenhancing cell viability.

FIGS. 2(a) to 2(e) illustrate in vitro MTT assay results using H112-2HIV viral strain¹⁹ which was not pretreated with AZT. As expected, theviral strain was sensitive to AZT. The data also showed that(-)-calanolide A was relatively non-toxic at concentrations of 1 μg/mLbut exhibited very little antiviral effect. (±)-Calanolide A was nearlyas effective as (+)-calanolide A in reducing viral CPE.

FIGS. 3(a) to 3(e) illustrate in vitro MTT assay results using A-17 HIVviral strain²⁰ which is resistant to to non-nucleoside inhibitors suchas TIBO but is sensitive to AZT. The results here parallel those shownin FIGS. 2(a)-2(e).

FIGS. 4(a)-(d) and 5(a)-(d) illustrate in vitro MTT assay results usinglab cultivated HIV viral strains IIIB and RF, respectively. The resultshere also parallel those shown in FIGS. 2(a)-2(e).

EXAMPLE 38 IN VITRO EVALUATION OF CALANOLIDE ANALOGUES

Selected calanolide A intermediates and analogues, prepared as describedabove, were evaluated using the in vitro MTT-tetrazolium assay describedin Example 37. As shown in the Table below, compounds (±)-7, (+)-7,(±)-8a, (±)-8b, (+)-10, (±)-16b, (±)-16d, (±)-16e, and (±)-16f werehighly efficacious in protecting cells against HIV infection.

                  TABLE                                                           ______________________________________                                        In Vitro Anti-HIV-1                                                             Activity of Analogues.sup.a                                                                Maximum                                                           Protection EC.sub.50 IC.sub.50                                               Compound (%) (μM) (μM) TI.sup.b                                       ______________________________________                                        (±)-7   90       1.18       19.10  16                                        (+)-7 90 0.68 72.80 107                                                       (±)-7a 55 2.82 12.00 4                                                     (±)-8a 84 6.16 23.8 4                                                      (±)-8b 81 2.28 21.50 9                                                     (+)-10 88 0.89 9.27 11                                                        (±)-14a c c 37.00 c                                                        (±)-14b c c 27.10 c                                                        (±)-15a c c 19.30 c                                                        (±)-15b c c 21.00 c                                                        (±)-15c 47 c 6.80 c                                                        (±)-16a c c 20.30 c                                                        (±)-16b 78 2.36 16.90 7                                                    (±)-16c c c 21.30 c                                                        (±)-16d 88 5.66 21.00 4                                                    (±)-16e 88 1.67 14.00 8                                                    (±)-16f 86 1.97 17.70 9                                                    (±)-16g c c 11.60 c                                                        (±)-16h c c 20.90 c                                                        (±)-18a 60 2.47 9.19 4                                                     (±)-19a c c 15.80 c                                                        (±)-19b c >100 >100 c                                                         22 c c 24.70 c                                                           ______________________________________                                         .sup.a CEMSS MTT assay                                                        .sup.b IC.sub.50 /EC.sub.50                                                   c not measurable                                                         

REFERENCES:

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(c) Shibata, I.; Baba, A., Organotin Enolates in Organic Synthesis. Org.Prep. Proc. Int. 1994, 26, 85-100.

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13. For a review on chiral boron complexes, see Paterson, L.; Goodman,J. M.; M., Aldol Reactions in Polypropinonate Synthesis: High π-FaceSelectivity of Enol Borinates from α-Chiral Methyl and Ethyl Ketonesunder Substrate Control. Tetrahedron Lett. 1989, 30, 7121-7124 andreferences cited therein.

14. Tsunoda, T.; Yamamiya, Y.; Kawamura, Y.; Ito, S., MitsunobuAcylation of Sterically Congested Secondary Alcohols by N, N, N¹, N¹-Tetramethylazodicarboxamide-Tributylphosphine Reagents. TetrahedronLett. 1995, 36, 2529-2530.

15. Crombie, L.; Jones, R. C. F.; Palmer, C. J., Synthesis of the MammeaCoumarin. Part 1. The Coumarin of the Mammea A, B, and C Series. J.Chem. Soc., Perkin Trans. 1, 1987, 317-331.

16. Very recently, a similar work has been published in the literature;Cardellina, J. H., II; Bokesch, H.

R.; McKee, T. C.; Boyd, M. R., Resolution and Comparative Anti-HIVEvaluation of the Enantiomers of Calanolides A and B. Bioorg. Med. Chem.Lett. 1995, 5, 1011-1014.

17. Deshpande, P. P., Tagliaferri, F.; Victory, S.F.; Yan, S.; Baker, D.C., Synthesis of Optically Active Calanolides A and B. J. Org. Chem.1995, 60, 2964-2965.

18. Gulakowski, R. J.; McMahon, J. B.; Staley, P. G.; Moran, R. A.;Boyd, M. R., A semiautomated Multiparameter Approach for Anti-HIV DrugScreening, J. Virol. Methods, 1991, 33, 87-100.

19. Larder, B. A.; Darby, G.; Richman, D. D., HIV with reducedSensitivity to Zidovudine (AZT) isolated during Prolonged Therapy,Science, 1989, 243, 1731-1734.

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What is claimed is:
 1. A compound of the formula II: ##STR7## wherein R₁is H, halogen, hydroxyl, amino, C₁₋₆ alkyl, aryl-C₁₋₆ alkyl, mono- orpoly-fluorinated C₁₋₆ alkyl, hydroxy-C₁₋₆ alkyl, C₁₋₆ alkoxy, amino-C₁₋₈alkyl, C₁₋₆ alkylamino, di(C₁₋₆ alkyl)amino, C₁₋₈ alkylamino-C₁₋₈ alkyl,di(C₁₋₆ alkyl)amino-C₁₋₈ alkyl, cyclohexyl, aryl or heterocycle, whereinaryl or heterocycle may each be unsubstituted or substituted with one ormore of the following: C₁₋₆ alkyl, C₁₋₆ alkoxy, hydroxy-C₁₋₄ alkyl,hydroxyl, amino, C₁₋₆ alkylamino, di(C₁₋₆ alkyl)amino, amino-C₁₋₈ alkyl,C₁₋₈ alkylamino-C₁₋₈ alkyl, di(C₁₋₆ alkyl)amino-C₁₋₈ alkyl, nitro, azidoor halogen;R₂ is H, halogen, hydroxyl, C₁₋₆ alkyl, aryl-C₁₋₆ alkyl,mono- or poly- fluorinated C₁₋₆ alkyl, aryl or heterocycle; R₃ and R₄are independently selected from the group consisting of H, halogen,hydroxyl, amino, C₁₋₆ alkyl, aryl-C₁₋₆ alkyl, mono- or poly- fluorinatedC₁₋₆ alkyl, hydroxy-C₁₋₆ alkyl, amino-C₁₋₈ alkyl, C₁₋₈ alkylamino-C₁₋₈alkyl, di(C₁₋₆ alkyl)amino-C₁₋₈ alkyl, cyclohexyl, aryl or heterocycle;and R₃ and R₄ can be taken together to form a 5-7 membered saturatedcycle ring or heterocyclic ring; R₅ and R₆ are independently selectedfrom the group consisting of H, C₁₋₆ alkyl, aryl-C₁₋₆ alkyl, mono- orpoly- fluorinated C₁₋₆ alkyl, aryl or heterocycle; and R₅ and R₆ can betaken together to form a 5-7 membered saturated cycle ring orheterocyclic ring; R₇ is H, halogen, methyl, or ethyl; R₈ and R₉ areindependently selected from the group consisting of H, halogen, C₁₋₆alkyl, aryl-C₁₋₆ alkyl, mono- or poly- fluorinated C₁₋₆ alkyl,hydroxy-C₁₋₆ alkyl, amino-C₁₋₈ alkyl, C₁₋₈ alkylamino-C₁₋₈ alkyl,di(C₁₋₆ alkyl)amino-C₁₋₈ alkyl, cyclohexyl, aryl or heterocycle; and R₈and R₉ can be taken together to form a 5-7 membered saturated cycle ringor heterocyclic ring; R₁₀ is H, acyl, P(O)(OH)₂, S(O)(OH)₂, CO(C₁₋₁₀alkyl)CO₂ H, (C₁₋₈ alkyl)CO₂ H, CO(C₁₋₁₀ alkyl)NR₁₁ R₁₂, (C₁₋₈ alkyl)NR₁₁ R₁₂ ; wherein R₁₁ and R₁₂ are independently selected from the groupconsisting of H, C₁₋₆ alkyl; and R₁₁ and R₁₂ can be taken together toform a 5-7 membered saturated heterocyclic ring containing saidnitrogen; or a pharmaceutically acceptable salt thereof, alone or incombination with a carrier.
 2. The compound of claim 1 wherein R₃ is H.3. The compound of claim 2 wherein R₄ is methyl.
 4. The compound ofclaim 3 wherein R₁₀ is H or Ac.
 5. The compound of claim 4 wherein R₈ isH.
 6. The compound of claim 5 wherein R₉ is methyl.
 7. The compound ofclaim 6 wherein R₁ is phenyl.
 8. The compound of claim 6 wherein R₁ isn-propyl.
 9. The compound of claim 1 wherein R₁ ═n-propyl; R₄ ═R₅ ═R₆═R₉ ═methyl; R₂ ═R₃ ═R₇ ═R₈ ═H; and R₁₀ ═H or Ac.
 10. The compound ofclaim 1 wherein R₁ ═n-propyl; R₃ ═R₅ ═R₆ ═R₈ ═methyl; R₂ ═R₄ ═R₇ ═R₉ ═H;and R₁₀ ═H or Ac.
 11. The compound of claim 1, R₁ ═n-propyl; R₄ ═R₅ ═R₆═R₈ ═methyl; R₂ ═R₃ ═R₇ ═R₉ ═H; and R₁₀ ═H or Ac.
 12. The compound ofclaim 1, R₁ ═n-propyl; R₃ ═R₅ ═R₆ ═R₉ ═methyl; R₂ ═R₄ ═R₇ ═R₈ ═H; andR₁₀ ═H or Ac.